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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
yes
Remarks:
but not relevant for the integrity of teh study
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Health check
Five male and 5 female rats were selected randomly after arrival, sacrificed and subjected to a full post mortem examination. Lungs, liver, kidneys, spleen, heart and any abnormalities were preserved in fixative and processed for pathological examination. All animals were considered healthy.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on mating procedure:
Matings were monogamous (one male to one female). Vaginal smears were taken, during pairing and up to the day of positive identification of mating 8for a maximum of 14 days for each pairing combination). Each cage was checked each morning for the presence of copulation plugs. The day of pairing combination is described as Day 0.
The pairing combinations were changed for the following female nos.: 81940001, 81940015, 81940023, 81940059, 81940069, 81940089, 81940151, 81940155, 81940169, 81940173.
The subsequent pairing was monitored for mating as described above for the first pairing. Mating was inadvertently not detected for female nos: 81940151, 81940155, 81940169 and 81940173.
The delivery date of female nos. 81940155 and 81940169 suggested the reckless mating with the first pairing combination.
At necropsy, after the completion of the mating period, the uteri of female nos. 81940151 and 81940173 were gravid and considering the development of foetuses, the copulation occurred with the second male selected for the pairing combination
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated in the range from 0.1 to 250 mg/ml. Linearity, accuracy and precision were within the limits stated in the RTC’s SOPs for suspensions (r > 0.98; accuracy 95-105%; precision CV < 5%).
Duration of treatment / exposure:
Males
Males are treated for at least 10 weeks prior to pairing and thereafter through the day prior to sacrifice.
Dose volumes will be calculated according to individual body weight on the first day of treatment and adjusted according to individual body weight at weekly intervals thereafter.
Females
Females are treated at least 2 weeks prior to pairing, during mating, gestation and post partum periods until the day before sacrifice (on post partum Day 20 or shortly after).
Dose volumes are calculated according to individual body weight on the first day of treatment and adjusted according to individual body weight at weekly intervals up to positive identification of mating and then according to body weight on Days 0, 6, 10 and 15 post coitum and on Days 1, 7 and 14 post partum.
Details on study schedule:
Group Treatment (mg/kg/day)+ Level
Number Males Females
1 0 0 Control
2 50 1 Low
3 250 5 Medium
4 1000 125 High
No. of animals per sex per dose:
24 females and 24 mnales

Examinations

Parental animals: Observations and examinations:
In vivo observations:
Mortality
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post mortem examinations to be carried out during the working period of that day. Female no. 81940145 was sacrificed for distocya. A complete necropsy was performed in all cases as detailed in section below.
Clinical signs
All clinical signs were recorded for individual animals.
At least once before commencement of treatment and once daily during treatment, each animal was observed and any clinical signs recorded. Animals were observed also on the day of despatch to necropsy.
Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.
During the pre-treatment period all animals were observed but data (negative with the exception of female no. 81940097, showing hairloss on muzzle) were not tabulated.
Body weight
Males were weighed on the day of allocation and then weekly from the first day of treatment to termination.
Females were weighed on the day of allocation and then weekly from the first day of treatment until positive identification of mating. After mating, the females were weighed on Days 0, 6, 9, 12, 15 and 20post coitum. Dams were weighed on Days 1, 4, 7, 14 and 21 post partum. One control female (no. 81940021) was inadvertently not weighed on post partum Day 1.
Food consumption
Food consumption was recorded weekly from allocation to pairing. For the females, during the post coitum period, food consumption was measured on Days 6, 13 and 20 post coitum starting from Day 0.
During the post partum period, food consumption was measured on Days 4, 7, 14 and 21 starting from Day 1.
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning starting from 2 weeks before pairing until a positive identification of mating was made. The vaginal smear data were examined to determine the following:
a) anomalies of the oestrous cycle;
b) pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Litter observations:
The total litter size (live and dead) was counted, sexed and examined for abnormalities as soon as possible after parturition was completed (Day 0/1 post partum). All live pups were individually identified on Day 0 post partum. All pups were weighed on Days 1, 4, 7, 14 and 21 post partum.
All litters were examined daily for dead or abnormal young and all pups found dead or sacrificed for humane reasons were necropsied.
Postmortem examinations (parental animals):
Organ weights
The organs listed in section table below from all animals completing the scheduled including Dam
81940067, test period were dissected free of fat and weighed.
The ratios of organ weight to body weight were calculated for each animal.
Tissues fixed and preserved
Samples of all the tissues listed in table below were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in Bouin's solution and all preserved in 70% ethyl alcohol).
Histopathological examination
The tissues required for histopathological examination are listed in table below. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. The examination was restricted as detailed below:
a) Tissues specified in table below from all animals in the control and high dose group killed at term.
b) Tissues specified in table below from all animals killed or dying during the treatment period.
c) All abnormalities in all groups.

Organs / Tissues Weight Fixation Microscopic
Preservation Examination
Abnormalities - ✓ ✓
Coagulating gland - ✓ ✓
Epididymides ✓ ✓ ✓
Ovaries ✓ ✓ ✓
Pituitary - ✓ ✓
Prostate ✓ ✓ ✓
Seminal vesicles ✓ ✓ ✓
Testes ✓ ✓ ✓
Uterus with cervix ✓ ✓ ✓
Vagina - ✓ ✓
Postmortem examinations (offspring):
Culled pups
Culled pups were subjected to an external examination. Sex confirmation was performed by gonadal inspection.
Decedent pups
Decedent pups were subjected to an external and internal examination. Sex confirmation was performed by gonadal inspection.
Pups at weaning
All pups were examined externally.
One pup/sex/litter (randomly selected) was examined internally. Sex confirmation was performed at necropsy by gonadal inspection.
Statistics:
For the body weights, body weight change, food consumption and organ weights the significance of the differences amongst group means was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error of variance. The homogeneity of the data was assessed by Bartlett’s Test before Dunnett’s Test was performed. If the data were found to be inhomogeneous, a modified t test (Cochran and Cox) was applied.
Statistical analysis of histopathological findings was carried out by means of the non- parametric Kolmogorov-Smirnov test.
The non-parametric Kruskal-Wallis analysis of variance was used for litter data, sex ratios, implantation and pre-birth loss data. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test.
The criterion for statistical significance was p<0.05.
The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Reproductive indices:
he following reproductive indices are calculated:
Males
Copulatory Index (%)
Fertility Index (%)
Females
Copulatory Index (%)
Fertility Index (%)
Males and females Copulatory Interval

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No relevant clinical signs were described during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant reduction in terminal body weight was observed in high dose males compared to controls. The differences noted in organ weights of the high dose males were considered to be due to the lower terminal body weight
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A statistically significant reduction in terminal body weight was observed in high dose males compared to controls. The differences noted in organ weights of the high dose males were considered to be due to the lower terminal body weight
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
he distribution of oestrus cycle was similar between groups.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Mortality: 
There were 3 unscheduled deaths during the study. One control male no. 81940032 died due to an accidental trauma on Day 39 of the study. In the high dose group female no. 81940145 was sacrificed on the day of delivery due to distocya (difficulty in parturition) and female no. 81940171 was found dead on Day 6 of gestation. 
The control male, macroscopically, did not show any pathological changes.
Female no. 81940145 did not show any relevant finding that could be considered treatment- related.
Female no. 81940171 showed, at necropsy examination red fluid in the thoracic cavity, adhesions between lung lobes and heart. At microscopic examination, marked pericarditis associated with acute inflammation in the pleura, alveolar haemorrhage with congestion in the lungs were also observed. Such changes were the factors contributory to death of this animal probably caused by a misdosing during oral administration.
Fate of females: 
A total of 7 females were found not pregnant at necropsy; 4 animals in the control group, 1 female each in the low dose, mid-dose and high dose groups. One high dose female was sacrificed for difficulty in parturition. Uterus with unilateral implantation was noted in one low dose and one high dose female. Mating was not detected in 4 high dose females. One low dose female had total litter loss within one day of parturition and one high dose female was found dead on gestation Day 6. 
The number of females with live pups on post partum Day 21 was 20 in the control, 22 in the low dose group, 23 in the mid-dose group and 19 in the high dose group.
Clinical signs: 
No relevant clinical signs were described during the study.
Body weights: 
High dose males had lower body weights compared to controls from Week 2 of study until sacrifice. This reduction was statistically significant from Week 7 until sacrifice.
Body weight of females was unaffected by treatment. 
A statistically significant reduction in body weight change was noted in high dose males from Week 2 until sacrifice and on Week 13 until sacrifice for mid-dose males.
A statistically significant reduction in body weight change was noted in mid-dose females on Day 15 of gestation and in high dose females on Day 20 of gestation. 
No differences in body weight change were noted in treated females during the post partum period when compared to controls.
Food consumption: 
Food consumption was unaffected by treatment both for males and females.
Oestrus cycle distribution: 
The distribution of oestrus cycle was similar between groups.
Reproductive parameters: 
No differences were seen between the treated and the control groups in the number of copulation plugs or in the pre-coital interval. The fertility and copulatory indices were similar between groups.
Implantation, pre-birth loss data and gestation length: 
No differences were observed between the control and the treated groups.
Terminal body weight: 
A statistically significant reduction in terminal body weight was observed in high dose males compared to controls.
Terminal body weight of females was unaffected by treatment.
Organ weights: 
The absolute prostate weight of the high dose group males was significantly lower than controls (at the statistical analysis). The relative testes weight was significantly increased compared to controls. These differences were considered to be due to the lower terminal body weight of animals and not related to treatment. 
Organ weights of females were unaffected by treatment.
Macroscopic observations: 
Unscheduled deaths 
Two high dose females and a control male died during the experimental phase of the study. Female no. 81940145, sacrificed for humane reasons on the Day of delivery, did not show at post mortem examination any relevant finding that could be considered treatment-related.
Female no. 81940171 which died on Day 6 of gestation, showed at post mortem examination red fluid in the thoracic cavity, adhesions between lung lobes (left and right cranial lobes) and heart.
Control male no. 81940032, died for accidental trauma on Day 39 of the study and macroscopically it did not show any finding.
Final sacrifice
Two treated males showed at post mortem examination reduced size of the testis and one epididymidis (male no. 81940122) or of the seminal vesicles and prostate (male no. 81940182); swelling and/or enlarged liver was noted in two high dose males.
Enlarged ovaries as well as red colour in the pituitary were noted in most treated and control females.
The above-mentioned changes were considered to be incidental and/or expression of spontaneous pathology or physiological changes related to oestrous cycle.
Microscopic observations
Unscheduled deaths
Female no. 81940145, as well as control male no. 81940032, did not show any pathological change in the tissue/organs examined.
Female no. 81940171 showed a marked pericarditis associated with acute inflammation in the pleura, alveolar haemorrhage with congestion in the lungs. Such changes were the factors contributory to death of this animal, probably caused by a misdosing during oral administration.
Final sacrifice
No treatment-related changes were seen in the males or in the females receiving 5,5 ́-Bis- 1H-tetrazole diammonium salt (BHT-2NH3).
The lesions reported in control and/or treated animals were considered to be either incidental in origin or expression of spontaneous pathology, often seen in this species under our experimental conditions.


Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Sex:
female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

pups were similar between groups.
Necropsy findings in decedent/culled and pups at weaning: 
Necropsy findings in pups did not show, at post mortem examination, any relevant finding that could be considered treatment-related.
Litter data: 
Litter data were unaffected by treatment.
Sex ratios: 
Sex ratios were unaffected by treatment.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
125 mg/kg bw/day (actual dose received)
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The reproduction toxicity of 5,5 ́-Bis-1H-tetrazole diammonium salt (BHT-2NH3), when administered once daily by oral gavage to Sprague Dawley rats at dosages of 50, 250 and 1000 mg/kg/day for males and 1, 5 and 125 mg/kg/day for females, was investigated.
Males
Males were treated for at least 14 weeks (10 weeks prior to mating and during the mating period) until the day before scheduled sacrifice. During the in-life phase, body weight, clinical signs, food consumption, mating performance, pre-coital intervals and fertility index were evaluated.
At necropsy a detailed external and internal examination was performed.
A histological examination of the epididymides, coagulating glands, pituitary, prostate, seminal vesicles and testes was carried out in all males of control and high dose groups.
A reduction in body weight and body weight change was noted in high dose males from Week 2 of study to termination and in the mid-dose males from Week 13 to sacrifice, when compared to controls.
No signs of toxicological significance were observed in any other parameters investigated neither during the in-life phase nor during the macroscopic examination at any dose. No treatment-related abnormalities on histopathological examination of the reproductive organs performed in the high dose animals were described.
Females
Treatment of females was performed daily for at least 2 weeks prior to mating. For females with positive identification of mating, the treatment continued daily up to and including post partum Day 20.
During the in-life phase, body weight, clinical signs, food consumption, oestrus cycle distribution, mating performance, gestation length, outcome of parturition, litter data and sex ratios and clinical signs of the offspring were evaluated. Pre-coital intervals and fertility index were calculated.
At necropsy, a detailed external and internal examination was performed in dams and in selected pups.
The corpora lutea and implantations were counted at necropsy. Pre- and post-implantation losses were calculated.
A histopathological examination of the ovaries, pituitary, uterus with cervix and vagina was carried out in all females of control and high dose groups.
A statistically significant reduction in body weight change was noted in mid-dose females on Day 15 of gestation and in high dose females on Day 20 of gestation.
No signs of toxicological significance were observed in any other parameters investigated, neither during the in-life phase nor during the macroscopic examination at any dose. No treatment-related abnormalities on histopathological examination of the reproductive organs performed in the high dose animals were noted.
On the basis of the above results, the dosage of 1000 mg/kg/day was considered to be the NOAEL (No Observed Adverse Effect Level) for males and the dosage of 125 mg/kg/day the NOAEL for females.
Executive summary:

In a one-generation reproduction study the test substance was administered to Sprague Dawley rats (24 males and 24 femeles per dosage group, 4 groups) by gavage at dose levels of 0, 50, 250 and 1000 mg/kg bw/day (males) resp. 0, 1, 5 and 125 mg/kg bw/day (females). The mean litter size was 15.3 - 16.55 per group, the size and the vuiability was not affected by the treatment.

With respect to toxicity on reproduction no treatment related effectes were observed. The NOAEL is stated with 1.000 mg/kg for males and 125 mg/kg bw/day for females.

The body weight of high dose males was statistically significant reduced compared to controls from Week 2 of study to sacrifice. Body weight of females was unaffected by treatment.

A statistically significant reduction in body weight change was noted in high dose males from Week 2 until sacrifice and on Week 13 until sacrifice for mid-dose males. A statistically significant reduction in body weight change was noted in mid-dose females on Day 15 of gestation and in high dose females on Day 20 of gestation.

Since the latter effect on body weight gain of females are not clearly dose depending, they are considered to be not releant and the NOAEL with respect to systemic toxicity is stated with 125 mg/kg bw for both sexes. This statement is conclusive, because also in the 28 d study on rats (according to OECD guideline 407) no statistically significant effects on body weight were detected up to 1,000 mg/kg bw per day after oral application.This study is acceptable and satisfies the guideline requirement for a one-generation reproductive study according to OECD 415 in rats.