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Toxicological information

Toxicity to reproduction

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Administrative data

screening for reproductive / developmental toxicity
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, acceptable for assessment.
Reason / purpose:
reference to same study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Staatstoezicht op de Volkgezondheid, 19 April 1989; 6 June 1991

Test material

Details on test material:
- Aqueous solution containing ca. 70wt% TBHP (CAS No. 75-91-2)
- Lot/batch No.: 920602/1991

Test animals

Details on test animals and environmental conditions:
- Source: Charles River Wigd GmbH, Sulzfeld, Germany
- Age at study initiation: approx. 9-10 wk
- Weight at study initiation: male = 311.1 g; female = 198.5 g
- Housing: individually (during premating, gestation and lactation) or in pairs of one male and one female (during mating)
- Diet (e.g. ad libitum): cereal-based stock diet ad libitum
- Water (e.g. ad libitum): tap-water ad libitum

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
Solutions of test substance were prepared in Millipore-filtered water and stored in brown glass bottles, in a refrigerator, until use.

Concentration in dosing solutions (mg Aq. TBHP-70/ml):
Main study: 0, 0.3, 1, 3

Dosing volume: 10 ml/kg bwt

The test substance was administered once daily by oral gavage (10 ml/kg) with the dose volume were adjusted for changes in body weight. Four batches of dosing solutions were prepared for the main study (on June 30, July 14 and 28, and August 11, 1992).
The main study commenced on 1 July 1992 (= nominal day 0) and was terminated on 10-14 August 1992 and the animals sacrificed a few hours after the last dose.

Animals received treatment for up to 45 consecutive days.
Details on mating procedure:
Following a 2 wk premating treatment period, one male was paired with one female of the same treatment in the male home cage. Females were checked the following morning for evidence of mating (either the presence of a vaginal plug or the presence of sperm in a vaginal smear). Females that had not mated were rehoused with the same male and the process repeated for up to one week. The day of detection of a vaginal plug or sperm in vaginal lavage was designated day 0 of pregnancy. Females were returned to their home cage after mating.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Dosing solutions were analysed by GC-FID using a Perkin Elmer 8500 gas chromatograph with fused silica 50 m x 0.21 mm column and 0.5 um film methylsilicon PONA. Stability was determined at room temperature for 3 days or in a refrigerator for up to 16 days. Homogeneity was checked by analysing samples from the top, middle and bottom of the dosing solutions. Results demonstrated that achieved concentrations were nominal.
Duration of treatment / exposure:
Main study: 41-45 days (in-life phase 1 July 1992 to 10-14 August 1992)
Frequency of treatment:
Details on study schedule:
Treatment commenced: 1 July 1992
Mating commenced: 15 July 1992
In-life phase ended: 14 August 1992
Doses / concentrationsopen allclose all
Doses / Concentrations:
0, 3, 10 or 30 mg/kg bwt/d
nominal in water
Doses / Concentrations:
2.1, 7 or 21 mg/kg bwt/d
other: actual dose received
No. of animals per sex per dose:
12 males, 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
The test substance was administered once daily by oral gavage for up to 45 consecutive days.
Positive control:
not relevant


Parental animals: Observations and examinations:
See Chapter 7.5.1, record Repeated dose toxicity: oral.001
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
The following observations were recorded on PND1 and PND4:
- litter size, number of live and dead pups
- liver litter weight
- number of males and females
- number with external abnormalities
Postmortem examinations (parental animals):
Necropsy and histopathology procedures are described in Chapter 7.5.1, record Repeated dose toxicity: oral.001
Postmortem examinations (offspring):
Surviving pups were sacrificed (ether) on PND4 and subject to macroscopic examination
Pregnancy, litter and survival data along with clinical and macroscopic findings were analysed using Fisher's exact probability test. Time to mating, duration of gestation and litter size were analysed by Kruskal-Wallis ANOVA followed by the Mann-Whitney U-test, and pup body weights by ANOVA followed by Dunnett's multiple comparison tests.
Reproductive indices:
The following reproductive indices were calculated:
- mating index
- fertility index
- fecundity index
- gestation index
- sex ratio
Offspring viability indices:
The following offspring viability indices were calculated:
- live birth index
- viability index

Results and discussion

Results: P0 (first parental animals)

Reproductive function / performance (P0)

Reproductive performance:
no effects observed

Details on results (P0)

Gross pathology and histopathology
Results from range-finding studies preliminary to this investigation revealed the presence of moderate to severe submucosal oedema in the stomach wall of rats given doses of 107 mg/kg bwt/d for 5 consecutive days; no effects at 30 mg/kg bwt/d. Other information regarding findings in F0 parental animals from this study is reported in Chapter 7.5.1.

Reproductive performance
There were no treatment-related differences in:
- precoital time
- the number of pregnant females
- duration of gestation
- mean numbers of corpora lutae
- number of total, pre-implantation and post-implantation sites

Female fertility and fecundity indices were unexpectedly low in the control, low and high dose groups. Re-examination of vaginal smears from these animals revealed no false-positives and the cause of these findings (considered unrelated to treatment by the study director) is unclear:

Selected results by dose level (0, 3, 10 or 30 mg/kg bwt/d):
- females mated: 11/12, 11/12, 11/11, 11/12
- females pregnant: 8/11, 5/11, 10/11. 7/11
- females with live pups: 8/8, 4/5, 10/10, 7/7
- mating index (%): 92, 92, 100, 92
- female fertility index (%): 67, 42, 91, 58
- female fecundity index (%): 73, 45, 91, 64
- gestation index (%): 100,100, 100, 100

Effect levels (P0)

open allclose all
Dose descriptor:
Effect level:
21 mg/kg bw/day (actual dose received)
Basis for effect level:
other: no adverse effects at highest dose tested
Basis for effect level:
other: Remark: toxicokinetic data demonstrate rapid conversion of TBHP to 2-methylpropan-2-ol (tert-butanol) in vivo, thereby minimising any potential for uptake and systemic distribution of unchanged TBHP following oral exposure.
Remarks on result:
not measured/tested
Effect level not specified Generation not specified (migrated information)

Results: F1 generation

General toxicity (F1)

Mortality / viability:
no mortality observed

Details on results (F1)

The mean number of pups per litter and sex ratio was comparable in all groups, however pup mortality between PND1 and PND4 appeared significantly increased in low and high dose litters but not in the intermediate dose group (i.e. no trend present):

Results by dose level (0, 3, 10 or 30 mg/kg bwt/d):
- mean pups/litter: 11,00, 10.75, 11.10, 11.57
- live birth index (%): 98.9, 100, 100, 100
- pup mortality day 1 (%): 1.1, 0.0, 0.0, 0.0
- pup mortality day 4 (%): 0.0, 26 ***, 0.9, 6.2*
- viability index day 1-4 (%): 100, 74, 99, 94

*p<0.05; *** p<0.001

Comment: The mortality recorded in the low dose group was strongly influenced by the loss of all 10 pups from one litter. This finding together with the absence of any dose-response relationship lead the study director to conclude that this was unrelated to treatment.
Comment: The finding of a significant increase in pup mortality in the high dose group (reflecting the death of 1 or 2 pups in 4 of 7 litters) was considered by the study director to be an artefact caused by the high survival of the control litters.

Effect levels (F1)

Dose descriptor:
Effect level:
21 mg/kg bw/day (actual dose received)
Basis for effect level:
other: no adverse effects at highest dose tested

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

The relevance of mortality present in the low and high dose groups from this study was considered in the TBHP risk assessment report (document R319_0710_HH dated November 2007; Chemical Substances Bureau, Bilthoven, the Netherlands), which noted:

No significant toxicological effects were observed on reproductive indices and litter data, except for pup mortality between day 1 and 4 post partum in the 3 and 30 mg/kg bw groups (control: 0/87 (0%), 3 mg/kg: 11/43 (26%) with one total litter loss of 10 pups, 10 mg/kg: 1/111 (1%) and 30 mg/kg: 5/81 (6%). The study authors stated that the loss of pups in the 3 mg/kg bw group was probably not toxicological relevant because these pups were largely from one litter, and because no increased mortality in the next higher dose group was observed. The higher pup mortality at 30 mg/kg could be treatment related according to the study authors. However, the incidence of postnatal death was considered to be very low and therefore of minor toxicological significance. No information on historic control data were provided to show that a 6% postnatal mortality is within the normal range. No toxicological relevant effects on body weight, clinical observations and macroscopical observations in the pups were observed. From these results it can be concluded that the NOAEL for reproductive/developmental toxicity in this study is 30 mg/kg bw 70% TBHP, the highest dose tested.

The findings were not considered sufficiently robust to support classification of TBHP as substance toxic to reproduction by the EU Classification and Labelling Work Group.

Applicant's summary and conclusion

No evidence of adverse effects on parental reproductive indices and litter data following repeated oral exposure.
Executive summary:

The reproductive toxicity of TBHP was investigated in a GLP-compliant guideline combined repeated dose and reproductive/developmental toxicity screening test (OECD 422)  in which groups of male and female Wistar rats (12/sex/dose level) received TBHP by oral gavage in water at nominal doses of 0, 3, 10 or 30 mg/kg bw/day for 45 consecutive days (actual received doses = 0, 2.1, 7 or 21 mg/kg bwt/day). Treatment commenced 2 weeks prior to mating. Dose levels were based on results obtained from two preliminary range-finding studies which demonstrated moderate to severe damage to the stomach lining at higher treatment levels. There were no significant treatment-related toxicological effects observed on parental reproductive indices and litter data, with a NOAEL of 21 mg TBHP /kg bwt/d (actual dose received).