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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on the results in the one-generation study, the definitive parental, reproductive and developmental No Observed Adverse Effect Level (NOAEL) was established as being at least 1000 mg/kg body weight/day.

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 August 2008- 22 December 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study performed in compliance with GLP, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to guideline
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.34 (One-Generation Reproduction Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): TBzTD
- Molecular formula: C30H28N2S4
- Molecular weight: 544.821
- Physical state: Off-white powder
- Stability under test conditions: Stable.
- Storage condition of test material: At room temperature in the dark
- Analytical purity: 97.7%
- Lot/batch No.: 80600108
- Expiration date of the lot/batch: 01 June 2010
Species:
rat
Strain:
other: Rat: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Males (P) 5-6xweeks and females (P) 12-13 weeks.
- Weight at study initiation: (P) Males: x-x g; Females: x-x g
- Fasting period before study: not applicable
- Housing:
Pre-mating Animals were housed in groups of 4 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 4 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation Offspring was kept with the dam until termination.
General Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap-water.
Analysis of bedding, paper, diet and water did not reveal any findings that were considered to have affected study integrity.
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C (actual range: 18.2 – 22.6 °C)
- Humidity (%): 30 – 70% (actual range: 31 - 94 %)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day.

IN-LIFE DATES:
From: 15 August 2008
To: 22 December 2008
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. From Week 3 onwards, formulations were prepared weekly. Adjustment was made for specific gravity of the vehicle (factor: 1.036).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX.
- Concentration in vehicle: 0, 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5mL/kg
Details on mating procedure:
Following a minimum of 70 days of exposure for the males and 14 days of exposure for the females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates). Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of a intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating has occurred, the males and females were separated. A maximum of 15 days was allowed for mating. If no evidence of copulation was obtained after 10 days, the female was placed with another male (for an additional five days) of the same treatment group who had successfully mated, again avoiding sibling mating. This was done for Female 159 of Group 3.

- M/F ratio per cage: 1/1
- Length of cohabitation: 15 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear; referred to as day 0 of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon cages (MIII type, height 18 cm).
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed according to a validated method (NOTOX Project 488598) in Week 1 and 16 of study.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115 %. Homogeneity was demonstrated if the coefficient of variation was ≤ 10 %. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

RESULTS:
Accuracy of preparation: The concentrations analysed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). In the chromatogram of one of the samples from the Group 1 formulation prepared for use in Week 16, a small response was observed at the retention time of the test substance. It was assumed to be introduced during analysis and not to derive from the formulation since the response was not observed in the duplicate test sample. In all other formulations of Group 1, no test substance was detected. Therefore, this finding was considered not to have affected the study integrity.

Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Stability: Analysis of Group 2 and Group 4 formulations after storage for 8 days in the refrigerator yielded a relative difference of +16% and +19% compared to t=0, respectively. Because the concentration had not decreased (but increased) during storage and because mean concentration after storage was 107 and 108% relative to target, the formulations were considered stable during storage in a refrigerator for at least 8 days.
Duration of treatment / exposure:
F0-generation:
Males: A minimum of 70 days prior to mating and continuing until the day before scheduled necropsy.
Females: A minimum of 14 days prior to mating and continuing until the day before scheduled necropsy. Animal No. 169 was not dosed on Day 1 of of lactation because of littering.

F1-generation: The F1-generation was potentially exposed to the test substance in utero and through nursing during lactation.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.
Details on study schedule:
- Age at mating of the mated animals in the study: 15-16 weeks (P)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Fo-parental animals: 24 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were selected based on results of a 28-day toxicity study (RCC NOTOX Project 0808/1474 (001654)). During the 28-day study, Sprague Dawley rats were treated at 0, 50, 200 or 1000 mg/kg body weight/day. No toxicity was observed up to 1000 mg/kg. Therefore, a no effect level (NOEL) was established at 1000 mg/kg body weight/day.
Positive control:
not applicable.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
Mortality / Viability: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
Clinical signs: At least once daily, detailed clinical observations were made in all animals. The time of onset, degree and duration was recorded.
Cage debris of pregnant females will be examined to detect abortion or premature birth, if applicable. Signs of difficult or prolonged parturition will be recorded, if applicable.

BODY WEIGHT: Yes
Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1, 4, 7, 14 and 21.

FOOD CONSUMPTION: Yes
Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1, 4, 7, 14 and 21 of lactation.

WATER CONSUMPTION:
Subjective appraisal was maintained during the study period but no quantitative investigation introduced as no effect was suspected.
- Time schedule for examinations:
Oestrous cyclicity (parental animals):
not applicable.
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations:
testis weight, epididymis weight, histopathology
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
Number of pups, stillbirths, live births
Mortality / Viability: The numbers of live and dead pups at the First Litter Check (= check at Day 1 of lactation) and daily thereafter were determined. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made in all animals.
Body weights: Live pups were weighed during lactation on Days 1, 4, 7, 14 and 21.
Sex: Was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS:
- Pups, younger than 7 days, will be killed by decapitation. All remaining pups will be sacrificed using an oxygen/carbon dioxide procedure.
- Culling will be performed on Day 4 of lactation or shortly thereafter.
- The remaining pups will be killed at Day 21 post-partum or shortly thereafter.

Offspring found dead or killed before Day 14 of lactation will be sexed and externally examined (if practically possible) with emphasis on developmental morphology. The stomach will be examined for the presence of milk.
Offspring found dead or killed on or after Day 14 of lactation will be sexed and subjected to external examination of the cranium, and macroscopic examination of the thoracic and abdominal tissues and organs (according to SOP/HIS/D/305), with emphasis on developmental morphology. Descriptions of all macroscopic abnormalities will be recorded.
If possible, defects or cause of death will be evaluated. Any abnormal pup will be preserved in 10% buffered formalin, bouin (Klinipath, Duiven, The Netherlands) or 96% ethanol, as appropriate, for possible further examination. All gross lesions will be fixed in 10% buffered formalin.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals; Males were killed as soon as possible after delivery of the litters.
- Maternal animals: All surviving animals; Females were killed on Day 21 post-partum or shortly thereafter.
- Females showing no evidence of copulation were killed approximately 21 days after the last day of the mating period.
- Non-pregnant females were killed on Day 25-27 post-coitum. In case a female was not pregnant, the uterus was stained using the Salewski technique (Salewski, 1964) in order to determine any former implantation sites (Salewski staining prepared at NOTOX using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

GROSS NECROPSY
After sacrifice all animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

ORGAN WEIGHTS
The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy:
- Epididymides
- Seminal vesicles
- Ovaries
- Testes
- Pituitary gland*
- Uterus
- Prostate gland*
* weighed when fixed for at least 24 hours

HISTOPATHOLOGY
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):
- Identification marks: not processed
- Prostate gland
- Cervix
- Seminal vesicles
- Coagulation gland
- Testes*
- Epididymides*
- Uterus
- Ovaries
- Vagina
- Pituitary gland
- All gross lesions

* Testes and epididymis were fixed in modified Davidson's solution (prepared at NOTOX using Formaldehyde
37-40%, Ethanol, Acetic acid (glacial)(all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.

The following slides were examined by a pathologist:
-The preserved organs and tissues of 10 selected animals/sex of Groups 1 and 4:
Group 1 Males 2, 7, 9, 13, 14, 16, 18, 19, 21, 23.
Females 97, 101, 102, 105, 108, 109, 111, 113, 117, 118.
Group 4 Males 76, 79, 81, 82, 84, 87, 89, 90, 93, 96
Females 170, 171, 174, 177, 181, 183, 186, 187, 189, 191.
-All gross lesions of all animals (all dose groups).
-The reproductive organs of all animals that failed to mate, conceive, sire or deliver healthy offspring
Group 1 Male 24 and Female 120.
Group 2 Males 41 and 48 and Females 137 and 144.
Group 3 Male 63 and Female 159.
Group 4 None.

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.


Postmortem examinations (offspring):
SACRIFICE
- Pups, younger than 7 days, were killed by decapitation. All remaining pups were sacrificed using an oxygen/carbon dioxide procedure.
- Culling was performed on Day 4 of lactation or shortly thereafter.
- The remaining surviving pups were killed at Day 21 post-partum or shortly thereafter.

GROSS NECROPSY:
F1- Animals were subjected to postmortem examinations (macroscopic examination) as follows:
Offspring found dead or killed before Day 14 of lactation were sexed and externally examined (if practically possible) with emphasis on developmental morphology. The stomach was examined for the presence of milk.
Offspring found dead or killed on or after Day 14 of lactation were sexed and subjected to external examination of the cranium, and macroscopic examination of the thoracic and abdominal tissues and organs, with emphasis on developmental morphology. Descriptions of all macroscopic abnormalities were recorded.
If possible, defects or cause of death were evaluated. Any abnormal pup was preserved in 10% buffered formalin, bouin (Klinipath, Duiven, The Netherlands) or 96% ethanol, as appropriate, for possible further examination. All gross lesions were fixed in 10% buffered formalin.

HISTOPATHOLOGY / ORGAN WEIGTHS
not applicable.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher 1950) was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Reproductive indices:
Percentage mating: Number of females mated/Number of females paired x 100
Fertility index: Number of pregnant females/Number of females paired x 100
Conception rate: Number of pregnant females/Number of females mated x 100
Gestation index: Number of females bearing live pups/Number of pregnant females x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
Percentage of postnatal loss Days 0-4 post-partum: Number of dead pups on Day 4 post-partum/Number of live pups at First Litter Check x 100
Percentage of breeding loss Day 5 until weaning: Number of dead pups between Days 5 and 21 post-partum/Number of live pups on Day 4 post-partum x 100
Percentage live males at weaning: Number of live male pups on Day 21 post-partum/Number of live pups on Day 21 post-partum x 100
Percentage live females at weaning: Number of live female pups on Day 21 post-partum/Number of live pups on Day 21 post-partum x 100
Viability index: Number of live pups on Day 4 post-partum/Number of pups born alive x 100
Weaning index: Number of live pups on Day 21 post-partum/Number of live pups on Day 4 post-partum x 100
Clinical signs:
no effects observed
Description (incidence and severity):
Pale faces was noted in males treated at 1000 mg/kg from Week 13 of treatment onwards and in females treated at 1000 mg/kg from Week 5 of treatment onwards. In absence of any corroborative findings, this finding was considered to be non-adverse.
Slight salivation was noted among the dose groups (males only). This was considered to be a physiological response to the taste of the formulation rather than a sign of systemic toxicity. Considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing), this was considered not toxicologically relevant.
Mortality:
no mortality observed
Description (incidence):
No treatment-related mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the study period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after allowance for body weight was similar between treated and control animals.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment related histopathological changes were observed in the tissues of any animal receiving TBzTD. No significant pathological findings were present in the reproductive organs of male and female animals that failed to produce offspring.
All microscopic findings were considered to be incidental and unrelated to treatment with the test substance.
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No treatment related effects in the organs examined.
Reproductive performance:
no effects observed
Description (incidence and severity):
No toxicologically significant effects on reproductive parameters were noted.
Percentage mating, fertility index, conception rate, pre-coital time, and number of corpora lutea and implantation sites of all dose groups were well within the normal range of variation in rats of this age and strain and were unaffected by treatment.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related effects. Incidental clinical symptoms of pups consisted of small and pale appearance, no milk in the stomach, dehydration, blue discoloration of the eyes, swelling and red discoloration of the ear, red spots on the nose or head, blue spots on the head or nose, wound on the back, cold, lean appearance, and alopecia and scabs at several body parts.
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment-related effects.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related effects.
Macroscopic examination of the pups revealed small appearance, no milk in the stomach and pelvic dilation of the kidney. For dead pups, autolysis was noted. No relationship with treatment was established for these observations or they were considered to be within the normal biological variation for rats of this age and strain.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
VIABILITY (OFFSPRING):
No treatment-related effects.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Highest dose tested. No substance related effects were observed
Reproductive effects observed:
not specified
Conclusions:
Based on the results in the one-generation study, the definitive parental, reproductive and developmental No Observed Adverse Effect Level (NOAEL) was established as being at least 1000 mg/kg body weight/day.
Executive summary:

Accuracy, homogeneity and stability of the formulations were demonstrated by analyses.

Parental results:

No unscheduled deaths occurred.

 

Treatment-related clinical signs comprised pale faeces in males and females treated at 1000 mg/kg during the last weeks of treatment. In absence of any corroborative findings, this finding was considered to be non-adverse.

 

No adverse effects on body weight, body weight gain, food consumption, macroscopy or organ weights were noted following treatment with TBzTD up to 1000 mg/kg.

 

At histopathological examination, morphologic alterations indicative of toxicity or alterations responsible for suspected infertility in the F0-generationwere not in evidence up to 1000 mg/kg.

Reproductive results:

Reproduction parameters(percentage mating, fertility index, conception rate, precoital time, and number of corpora lutea and implantation sites) werenot affected by treatment with TBzTDup to 1000 mg/kg.

 

Developmental results:

Developmental parameters (gestation index, duration of gestation, number of dead and living pups at first litter check, sex ratio, postnatal loss, viability index, and earlypostnatalpup development) werenot affected by treatment with TBzTDup to 1000 mg/kg.

 

CONCLUSION

Based on the results in this one-generation study, the definitive parental, reproductive and developmental No Observed Adverse Effect Level (NOAEL) was established as being at least 1000 mg/kg body weight/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Quality of whole database:
GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

An OECD Guideline 415 One-Generation Reproduction Toxicity Study was performed in compliance with GLP. The test substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 0, 100, 300 and 1000 mg/kg bw/day (24 rats/sex/dose level) dissolved in propylene glycol. Accuracy, homogeneity and stability of formulations were demonstrated by analyses. Following a minimum of 70 days of exposure for the males and 14 days of exposure for the females, one female was cohabitated with one male of the same treatment group. Males were killed as soon as possible after delivery of the litters and females were killed on Day 21 post-partum or shortly thereafter. Females showing no evidence of copulation were killed approximately 21 days after the last day of the mating period and non-pregnant females were killed on Day 25-27 post-coitum. In the parents, no unscheduled deaths occurred. Treatment-related clinical signs comprised pale faeces in males and females treated at 1000 mg/kg during the last weeks of treatment. In absence of any corroborative findings, this finding was considered to be non-adverse. No adverse effects on body weight, body weight gain, food consumption, macroscopy or organ weights were noted following treatment with TBzTD up to 1000 mg/kg. At histopathological examination, morphologic alterations indicative of toxicity or alterations responsible for suspected infertility in the F0-generation were not in evidence up to 1000 mg/kg. Reproduction parameters (percentage mating, fertility index, conception rate, precoital time, and number of corpora lutea and implantation sites) were not affected by treatment with TBzTDup to 1000 mg/kg. Developmental parameters (gestation index, duration of gestation, number of dead and living pups at first litter check, sex ratio, postnatal loss, viability index, and early postnatal pup development) were not affected by treatment with TBzTD up to 1000 mg/kg. Based on the results in this one-generation study, the definitive parental, reproductive and developmental No Observed Adverse Effect Level (NOAEL) was established as being at least 1000 mg/kg body weight/day.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results of the one-generation study, the substance does not need to be classified according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Additional information