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Annex 2: Study design and results of the NTP main study

Study design

The group size at the beginning of the main study was 105 animals per exposure level and control group for both species (mice and rats) for GSM and CDMA modulation (exception: here a common control group) and for both sexes. Almost 1900 rats and 1500 mice were used in the study. In rats, the mothers were exposed from the fifth day of gestation; the mice were exposed from the age of 5–6 weeks.

The freely moving animals were kept individually under well-known laboratory conditions, which differed only in exposure; the control group was sham exposed. Any concomitant effects of exposure (e.g. sounds) were identified and adjusted in the experiment between the exposure groups in order to ensure good comparability between the exposed and non-exposed animals. The intermittent whole body exposure (10 minutes on/10 minutes off) of the animals was 18 h and 20 min daily; this resulted in a total daily exposure duration of 9 h and 10 min.

Intermediate analysis

An intermediate analysis was performed 14 weeks after the start of the study. Ten female and ten male rats and mice per group were euthanised, dissected, and examined for different endpoints (e.g. organ weights, microscopic examination of main tissues, sperm mobility and concentration, and vaginal cytology). In order to investigate genotoxicity, blood and tissue samples were taken from five male and five female rats and mice per group.

End of study

This left 90 animals per chronically long-term exposed exposure group and control group. After two years (roughly corresponding to the average life expectancy of rats and mice), any animals still living were euthanised. All animals, including those that were found dead during the study or had to be euthanised because of illness, were dissected and examined.

Statistical tests

To test whether sham-exposed and exposed animals differ in terms of possible long-term effects, two types of statistical tests (separate for mice and rats, male and female animals, and GSM and CDMA modulation) were performed for the aforementioned endpoints (intermediate analysis and end of study). In the first test, one of the three exposure groups was compared with the control group (pairwise comparison). In the second test, it was examined whether there was a trend with increasing intensity of exposure. The significance level for the statistical tests was 0.05 (pathology).

Results

Investigations in ratsShow / Hide

1. Results of exposure of mothers
  • Body weights of the exposed mothers: No difference to sham-exposed control group. However, weight gain in the 6 W/kg GSM-exposed group was significantly reduced (by 7%) between day 6 through 21 after conception compared with the sham-exposed control group.
  • Litter size: No impairment by exposure.
  • Mortality or survival rate: No effects were observed in young animals exposed to GSM. In the rats exposed to 6 W/kg CDMA, a significantly increased mortality rate of the young animals was observed compared with the sham-exposed control animals between day 4 and 21 after birth.
  • Body weights during the lactation period: in mothers, significantly lower in the 3 and 6 W/kg CDMA- and GSM-exposed groups compared with the sham-exposed control group. Body weights of the female and male young animals were lower four days after birth (CDMA) or at most times (GSM) in the 3 W/kg and at all times in the 6 W/kg exposed group (CDMA and GSM) compared with the sham-exposed control group.
2. Results of the intermediate analysis
  • Organ weights: Liver and kidney showed significantly lower weights in some exposure groups (in 1.5 and 6 W/kg GSM- and 6 W/kg CDMA-exposed male rats and in 6 W/kg CDMA- and 1.5, 3, and 6 W/kg GSM-exposed female rats). However, these were not associated with histopathological findings.
  • Haematological and clinical-chemistry variables: Leukocyte and lymphocyte levels significantly decreased in female rats exposed to 3 W/kg GSM. Cholesterol and triglyceride levels were reduced in female rats exposed to 6 W/kg GSM. CDMA exposure showed no corresponding changes in haematological and clinical chemistry variables.
  • Reproductive organ weights; sperm motility or concentration: No effects from GSM or CDMA exposure.
  • Vaginal cytology: Because of the low diagnostic quality of the cytological sections, no evaluation was possible.
  • Cardiomyopathy in the right ventricle of the heart: Increased incidences in male rats of the 3 and 6 W/kg GSM-exposed and CDMA-exposed groups. Cardiomyopathy is a frequently observed spontaneous disease in rats and typically does not manifest clinically.
  • DNA damage: In the comet assay, significant increase in 6 W/kg CDMA-exposed males in hippocampus cells compared with sham-exposed control group. A significant positive trend was observed for DNA damage of hippocampus cells, cells of the frontal cortex, and blood leukocytes (by counting of 150 cells) of CDMA-exposed male rats.
3. Results of the two-year exposure of rats

  • Weight and weight gain and survival

    • The survival probability of all GSM- and CDMA-exposed groups was higher in male rats than in the sham-exposed control group. This effect was significant in the 1.5 and 3 W/kg CDMA-exposed group as well as in all GSM-exposed groups. The authors attribute the life-prolonging effect to the significantly reduced severity of chronic kidney disease (nephropathy) in exposed male rats. However, GSM-exposed female rats showed the same survival probability as female rats in the sham-exposed control group, while 6 W/kg CDMA-exposed female rats showed significantly higher survival.
    • The body weight of 6 W/kg GSM-exposed male rats was 3–6% lower up to day 401. From day 541 until the killing of the animals at the end of the study, the mean body weight was up to 7.2% higher. The body weight of the 6 W/kg CDMA-exposed male rats was lower than that of the sham-exposed animals until day 457. However, at the end of the study, the body weights of both groups were once again equal. In the females, there were no differences in body weight between exposed and sham-exposed animals in both modulations.

  • Neoplastic and non-neoplastic lesions of the heart, brain, and adrenal medulla have already been listed in the statement but are listed again here for completeness:

    • Heart: Increased incidence of malignant schwannomas in all GSM- (0/2/1/5) and CDMA- (0/2/3/6*) exposed groups of male rats. Both modulations show a significantly positive trend. In female rats, incidences increased only in the 3 W/kg GSM (GSM 0/0/2/0) and the 1.5 and 6 W/kg CDMA-exposed groups (0/2/0/2). Increased incidence of Schwann cell hyperplasia (possible preneoplastic Schwann cell lesion) in male (GSM, 0/1/0/2; CDMA 0/0/0/3) and female rats (CDMA 0/1/1/1 only).

      Increased incidences of right ventricular cardiomyopathies in all groups of male (GSM, 54/62/72*/74*; CDMA, 54/45/62/74*) and female rats (GSM, 4/9/14*/15*; CDMA, 4/7/9/9).

    • Brain: Increased incidence of malignant gliomas in all GSM-exposed groups of male rats (0/3/3/2) and in the 6 W/kg exposed group of female rats (0/0/0/1). For CDMA increased incidences in male (0/0/0/3, significantly positive trend) and female rats (0/3/0/0).

      Increased incidence of hyperplasia of glial cells (possible preneoplastic lesion that may develop into a malignant glioma) in most exposed groups of both sexes. However, this was not significant.

    • Adrenal medulla: Significantly increased incidences of benign pheochromocytomas (GSM: 10/23*/25*/14; CDMA, none) as well as benign, malignant, and complex pheochromocytomas in male rats (GSM: 11/24*/28*/14; CDMA, none). In female rats, slightly increased incidences of benign pheochromocytomas in all groups (GSM: 1/3/3/2; CDMA, 1/7/3/4) as well as increased incidences of benign, malignant, and complex pheochromocytomas in all CDMA-exposed groups (1/9*/5/4).

    • Other organs: Lesions in various organs (e.g. tumours of the prostate gland, pituitary gland, or pancreas) were observed. However, it was not possible to assess whether these resulted from exposure or were accidental findings. The exposure-dependent reduction of the severity of chronic nephropathy in male rats was reflected in an exposure-dependent reduction of the incidence of secondary lesions such as mineral deposits and chronic inflammations in blood vessels and hyperplasia of the parathyroid gland.

Investigations in miceShow / Hide

1. Results of the intermediate analysis
  • Haematological and clinical-chemistry variables: No changes that can be traced to the GSM or CDMA modulated exposure.
  • Organ weights: Significantly lower kidney and liver weights in the 5 and 10 W/kg GSM- and CDMA-exposed males compared with the sham-exposed control groups. However, the weight changes were negligible and were not accompanied by exposure-related histopathological lesions. Significantly lower brain and right kidney weights in females exposed to 10 W/kg GSM. These changes were considered non-toxicologically important. In CDMA-exposed females, there were no weight changes of the organs.
  • Reproductive organ weights; sperm motility or concentration: No effects from GSM or CDMA exposure in male mice.
  • Vaginal cytology: No effects in females.
  • Focal inflammation in the liver: Significantly increased incidence in the 5 W/kg GSM-exposed male mice and an insignificant increase in the 2.5 W/kg GSM-exposed group. However, focal inflammations are frequently observed in this mouse strain and were not considered biologically relevant in the study.
  • DNA damage: In the comet assay, significant increase in male mice in cells of the frontal cortex in both modulations (at 5 and 10 W/kg CDMA and at 10 W/kg GSM) compared with the sham-exposed control group as well as a significantly positive trend. Significantly increased DNA damage in blood leukocytes in CDMA-exposed female mice at all three exposure levels.
2. Results of the two-year exposure of mice

  • Weight and weight gain and survival

    • Survival probability: Significantly increased in 5 W/kg GSM-exposed and 2.5 W/kg CDMA-exposed male mice compared with sham-exposed control group. The survival probability of male and female mice of the other exposed groups was equal to that of the sham-exposed control groups.
    • Weight and weight gain did not differ between exposed and sham-exposed mice in both modulations.

  • Neoplastic and non-neoplastic lesions

    • Liver tumours: Significantly increased incidences of hepatocellular adenomas at 2.5 W/kg CDMA (52/66*/55/62) and hepatoblastomas at 5 W/kg CDMA (6/6/16*/7) in male mice.
    • Skin tumours: Increased but not significant at 5 and 10 W/kg GSM in male mice (1/1/5/4).
    • Lung tumours: Significant increase with increasing exposure in male mice with GSM (23/24/32/34).
    • Malignant lymphoma: Increases in all GSM- (2/13*/9*/6) and CDMA- (2/9*/6/7) exposed female mice.
    • Heart and brain were unremarkable.

The extraordinary transparency of the NTP is a positive aspect. A considerable amount of data (e.g. incidence tables) is freely available on the NTP servers to verify the validity of the statistical evaluation and others. The tissue sections can also be viewed in the archives of the NTP.

State of 2021.04.14

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