Tributylmethylammonium chloride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Two oral OECD 422 screening studies with MTBAC are available (Klimisch 1 studies). The parental and reproduction NOAEL was determined to be >= 1000 mg/kg bw/day in both studies.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 May 2015 - 28 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

(March 1996)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (July 2000)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD 421, Reproduction/Developmental Toxicity Screening Test (July 1995)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: See other Guidelines under "Principles of method if other than guideline"

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)

Deviations:

no

Principles of method if other than guideline:

In addition, the procedures in this study essentially conformed to the following guidelines:

Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008);
OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008):
The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: 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: Approximately 10-12 weeks
- Weight at study initiation: 295-331 g (males); 207-231 g (females)
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages;
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages;
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages;
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany); All males and the selected 5 females/group were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy
- Water: Free access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 18 – 24 (temporary deviations from the daily mean relative humidity occurred, but laboratory historical data do not indicate an effect of the deviations)
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 May 2015 to 28 August 2015

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Method of formulation: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance and vehicle. No correction was made for the purity/composition of the test substance.

Storage conditions of formulations: At room temperature protected from light.

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Details on mating procedure:

- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dose preparations were taken at the test facility on a single occasion during the treatment period (formulations were prepared and sampled on 06 July 2015). The samples were dispatched on dry ice to second site where they were analyzed to assess accuracy of preparation (all groups), homogeneity (lowest and highest concentration) and stability in vehicle over 5 hours at room temperature protected from light (lowest and highest concentration).

Duration of treatment / exposure:

Males were exposed for 31 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Pups were not treated directly, but were potentially exposed to the test substance in utero, through lactational transfer or via exposure to maternal urine or faeces.

Frequency of treatment:

Once daily for 7 d/w.

Details on study schedule:

- Age at mating of the animals in the study: Approximately 13 weeks

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:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were based on results of a 10-Day dose range finding study, in which 3 females per group were exposed to 500 or 1000 mg/ kg bw..
No mortality occurred in this study. At 500 mg/ kg bw/ day, all 3 rats showed salivation on 2-4 days. At 1000 mg/kg bw/ day, hunched posture was seen on 1-3 days for all 3 animals, flat gait on one day for 3 animals and salivation on 6-7 days for 3 animals. Furthermore, one animal showed piloerection on one day. Body weight gain was normal at 500 mg/ kg bw/ day, however at 1000 mg/ kg bw/ day 2 animals lost weight from day 1-5 and had slight weight gain from day 5-10. No effects were seen in either groups on food consumption and at macroscopic evaluation. Liver and kidney weights were normal for all treated animals.
Selection of animals for selected measurements (main study):
5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology. Only females with live offspring were selected. Histopathology was done on rats that died spontaneously (if possible).

Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.

Identification of pups:
On day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
Yes
- Time schedule: At least once daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT:
Yes
- Time schedule for examinations: 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 and 4.

FOOD CONSUMPTION:
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY:
Yes (Relative food consumption (g/kg body weight/day was calculated)

WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Yes
- Time schedule for examinations: The selected males were tested during week 4 of treatment and the selected females were tested during lactation (from lactation day 4 onwards).
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and locomotor activity (total movements and ambulations)

Oestrous cyclicity (parental animals):

Only determined at necropsy.

Sperm parameters (parental animals):

Slides of the testes were prepared for histopathological staging of spermatogenesis (5 males of the control and high dose group, and for all males that did not reproduce successfully).

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on 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 on Days 1 and 4 of lactation.
- Sex: 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
Yes, if possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

GROSS PATHOLOGY
All animals were fasted overnight (with a maximum of 20 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.

The number of former implantation sites and corpora lutea were recorded for all paired females.

- Selected 5 animals/sex/group and all animals that died spontaneously or were killed in extremis: According to test guidelines

- All remaining animals and females which failed to deliver: According to test guidelines

ORGAN WEIGHTS
- Selected 5 animals/sex/group: According to test guidelines

- All remaining males:
Epididymides and Testes

HISTOPATHOLOGY
According to test guidelines

Postmortem examinations (offspring):

SACRIFICE
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
No.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 1; many-toone 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 (Ref. 2; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 3) was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test (Ref. 4) was applied to motor activity data to determine intergroup differences.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Ref. 1 Dunnett C.W., A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
Ref. 2 Miller R.G., Simultaneous Statistical Inference, Springer Verlag, New York (1981).
Ref. 4 Fisher R.A., Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
Ref. 5 Kruskal W.H. and Wallis W.A.. Use of ranks in one-criterion variance analysis. Journal of the American Statistical Association 47 (260): 583-621, December (1952).

Reproductive indices:

For each group, the following calculations were performed:
- Mating index (%): (Number of females mated/Number of females paired) x 100
- Fertility index(%): (Number of pregnant females/Number of females paired) x 100
- Conception index(%): (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 of lactation: (Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check) x 100
- Viability index (%): (Number of live pups on day 4 of lactation / Number of pups born alive) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were noted during the observation period. Salivation seen after dosing among some animals of the 300 mg/kg bw/ day group and all animals of the 1000 mg/kg bw/ day dose group during was considered to be a physiological response 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). Alopecia was noted in one control female and was not considered related to the treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female at 1000 mg/kg bw/ day was found dead on day 1 of the mating period and showed beginning autolysis at necropsy (cause of death unclear). No other mortality occurred.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No treatment-related changes in body weights and body weight gain were noted in any of the groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment-related changes in food consumption before or after allowance for body weight were noted up to and including 1000 mg/kg bw/ day.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes occurred in haematological parameters of treated rats. Incidental findings (increase in lymphocytes in males at 300 mg/kg bw/ day; increased platelet concentration in females at 100 mg/kg bw/ day) were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Several blood parameters were altered in males in the highest dose group (statistically significant): decreased total protein and albumin concentration, increased creatinine, and slightly increased potassium, chloride and inorganic phosphate levels. Reduced concentration of bile acids was observed in exposed male rats (reduction was statistically significant for mid and high dose group). It is of note that two control males were present with high bile acid concentration. As the values of exposed rats were still within the normal range, this effect was not considered toxicologically relevant. In parallel to males, also in the high dose females decreased total protein and albumin concentration was found (both statistically significant). Furthermore, statistically significant increase of aspartate aminotransferase (ASAT) activity was seen in this group. The level was however within the range considered normal for rats of this age and strain and the effect on ASAT was not considered to be toxicologically relevant. No other parameters were affected by the treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period. It was noted that the mean value for ambulations was lower in females at 1000 mg/kg bw/day, although not statistically significant. The difference was due to low values in 3/5 females and variation was high, also in the control group. Therefore, this difference was considered not to be related to treatment with the test substance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic findings. Minimal squamous cell hyperplasia of the forestomach, slight glandular erosion and slight hemorrhage and congestion of the glandular stomach (both correlated to several dark red foci of the glandular mucosa noted at necropsy) in the stomach of a single male of the 1000 mg/kg/day group. These findings were regarded to be caused by trauma during the gavage procedure and not directly related to the test item. Remaining recorded histologic changes were considered to be incidental findings. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No abnormalities were observed spermatogenesis of control and high dose group.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. There were two couples which failed to deliver healthy pups (one in control group and one in highest dose group (female died on first day of mating period)). No abnormalities were seen in the reproductive organs, spermatogenic profiles of both males were normal. Gestation index and duration of gestation were not affected by treatment. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen at highest dose tested.

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical symptoms were noted in any of the pups.

Mortality / viability:

no mortality observed

Description (incidence and severity):

Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were unaffected by treatment.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

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 abnormalities were noted in surviving pups. The pup that died at 300 mg/kg bw/ day showed beginning autolysis and had no milk in the stomach. The pup that died at 1000 mg/kg bw/ day had no abnormalities.

Histopathological findings:

not examined

Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related effects.

Dose descriptor:

NOAEL

Remarks:

reproduction

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects seen at highest dose tested.

Critical effects observed:

no

Reproductive effects observed:

not specified

No test substance was detected in the control group formulations. The concentrations analysed in the formulations of te test substance groups were in agreement with the target concentrations (i.e. mean accuracies between 98.5% and 99.1%). The formulations of the low and high dose group were homogeneous (i.e. coefficient of variation 0.6% and 1.1% respectively). Formulations at the entire range were stable when stored at room temperature protected from light for at least 6 hours (i.e. relative difference ≤ 0.8%). The long term storage samples were stable at ≤-70°C for 7 days (mean recovery≥93.7%).

Conclusions:

In an oral OECD 422 screening study, the parental and reproduction NOAEL was derived to be 1000 mg/kg bw/day.

Executive summary:

A sub-acute oral study with screening for developmental/ reproduction effects was performed according to OECD/EC guidelines and GLP principles with MTBAC. MTBAC was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 31 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41-56 days). Formulation analysis confirmed that formulations were accurately and homogenously prepared. The parental NOAEL was determined to be 1000 mg/ kg bw/ day, based on absence of adverse effects at 1000 mg/ kg bw/ day. One couple in control group and one couple in highest dose group did not reproduce (female in highest dose group died on day 1 of meeting, death not substance related). No spermatogenic abnormalities were found. Gestation index and duration of gestation were not affected by treatment. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed. The number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related effects.

Based on these data, the parental and reproduction No Observed Adverse Effect Levels (NOAEL) for MTBAC were found to be 1000 mg/kg bw/ day.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 December 2015 - 16 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

28 Jul 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

Jul 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AMTBC14001
- Expiration date of the lot/batch: 22 Aug 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature; no direct sunlight
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor.

Species:

rat

Strain:

other: Crl:WI(Han)

Details on species / strain selection:

The rat is the preferred animal species for reproduction studies according to the various test guidelines and the Wistar strain was selected. This Wistar rat strain (Crl:WI(Han)) was selected since extensive historical control data were available on these Wistar rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 11-12 weeks (male animals), about 10 weeks (female animals)
- Fasting period before study: no
- Housing: during pre-treatment in Polysulfonate cages Typ 2000P (TECHNIPLAST, Hohenpeißenberg, Germany); during pre-mating, mating, gestation, lactation, males after mating and females after weaning in Polycarbonate cages type III; for motor activity (MA) measurements the animals were housed individually in polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany
- Diet: ad libitum, ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum
- Acclimation period: 20 days

DETAILS OF FOOD AND WATER QUALITY: The supplier assayed the food used in the study for chemical and microbiological contaminants. The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring Department of BASF SE as well as for the presence of microorganisms by a contract laboratory.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water heated up to 60°C was filled up to the desired volume and subsequently released with a magnetic stirrer. The test substance preparations were produced weekly, at least.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: in Polycarbonate cages type III; Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At the beginning (during pre-mating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. These samples were used as a concentration control at the same time. At the time points mentioned above, additionally one sample from the mid concentration was taken for concentration control analysis.The samples collected at the beginning of the administration period and during the lactation period
were analyzed.

Duration of treatment / exposure:

The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.
females: 57 days
males: 29 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle control

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels for the present study were selected at the request of the sponsor.

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily, before the administration as well as within 2 hours and within 5 hours after the administration

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals
- Parameters: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: determined before start of administration period in order to randomize the animals, during the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day.
The following exceptions are notable for the female animals:
During the premating phase, body weight was determined twice a week, i.e. on study days 3, 7, 10 and 14.
During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
Females with litter were weighed on the day of parturition (PND 0), PNDs 4, 7, 10 and 13.
Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males.
Females without litter and after weaning (PND 13) were weighed once a week.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: generally once a week
Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
Food consumption of the females with evidence of sperm was determined for GD 7, 14 and 20.
Food consumption of the females which gave birth to a litter was determined for PNDs 4, 7, 10 and 13.

WATER CONSUMPTION: Yes
- Time schedule for examinations: generally once a week
Water consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
Water consumption of the females with evidence of sperm was determined for GD 7, 14 and 20.
Water consumption of the females which gave birth to a litter was determined for PNDs 4, 7, 10 and 13.
Water consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

Oestrous cyclicity (parental animals):

For all females of the pool estrous cycle normality was evaluated before the beginning of the administration. In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.

Sperm parameters (parental animals):

Parameters examined in male parental generation:
testis weight, epididymis weight, seminal vesicles with coagulating glands weight, glans penis weight, M. levator ani together with M. bulbocavernosus weight, prostate weight, bulbourethral gland (Cowper’s gland) weight, stages of spermatogenesis

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, sex ratio, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups; blood samples from the PND 13 pups assessed for serum levels for thyroid hormones (T4)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; Thyroid glands/parathyroid glands of one selected male and one female pup per litter; All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
All parental animals were sacrificed by decapitation under isoflurane anesthesia.

GROSS NECROPSY: Yes

HISTOPATHOLOGY / ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Bulbourethral gland (Cowper’s gland), Epididymides, Glans penis, M. levator ani together with M.bulbocavernosus, Prostate, Seminal vesicles with coagulating glands, Testes, Thyroid glands (fixed)
The following weights were determined in 5 animals/sex and test group sacrificed on schedule (females with litters, same animals as used for clinical pathology examinations): Adrenal glands, Brain,Heart, Kidneys, Liver, Spleen, Thymus

The following organs or tissues of all parental animals were fixed in in 4% neutral-buffered formaldehyde or in modified Davidson’s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow(femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Eyes with optic nerve, Esophagus, Extraorbital lacrimal glands, Epididymides (modified Davidson’s solution), Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Target organs, Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus (uteri of all cohabited female F0 parental animals were stained according to Salewski E (1964)), Vagina

Postmortem examinations (offspring):

GROSS NECROPSY
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and the organs were assessed macroscopically.
On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution.
All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

Statistics:

Blood parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with control group was performed using WILCOXON-test for hypothesis of equal medians
Water consumption, food consumption, body weight, body weight change, gestation days, anogenital distance, anogenital index: Simultaneous comparison of all dose groups with the control group using the DUNNETT test for the hypothesis of equal means
Mating indices, fertility indices, females mated, females delivering, gestation index, females with stillborn pups, females with all stillborn pups: Pair-wise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions
Mating days until day 0 p.c., % postimplantation loss, pups stillborn, % perinatal loss, nipple development: Pair-wise comparison of dose group with control group using WILCOXON test with BONFERRONI-HOLM adjustment for the hypothesis of equal medians
Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index: Pair-wise comparison of dose group with control using WILCOXON test with BONFERRONI-HOLM adjustment for the hypothesis of equal medians
% live male day x, %live female day x: Comparison of dose group with control group was performed using WILCOXON test for hypothesis of equal medians.
Number of cycles, Cycle Length, Rearing, grip strength of fore limbs and hind limbs, landing foot-splay test, motor activity: Non-parametric one-way analysis using KRUSKAL-WALLIS test . If resulting pvalue was equal or less than 0.05, a pair-wise comparison of dose groups with control group was per
formed using the WILCOXON test for hypothesis of equal medians.
Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If resulting p-value was equal or less than 0.05, pairwise comparison ofeach dose group with control was performed using WILCOXON-test for equal medians

Reproductive indices:

Mating, fertility and gestation indices (only females) indices were calculated

Offspring viability indices:

The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters. The implantations were counted and the postimplantation loss (in %) was calculated.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Slight salivation shortly after test-substance administration was observed in animals of test group 3 (1000 mg/kg bw/d) as well as in 7 male and 7 female animals of test group 2 (300 mg/kg bw/d). At the same time, 4 male and 2 female animals of test group 3 (1000 mg/kg bw/d) ploughed their noses into bedding. From the temporary, short appearance immediately after dosing (or shortly before) it was concluded that both kinds of findings were induced by a bad taste of the test substance or local affection of the upper digestive tract. The effects were related to the test substance but assessed as being non-adverse as no significant lesions in the upper digestive tract were observed in male and female animals during pathological examinations. During the mating period, one female started to show piloerection. The finding was also present at later periods of the study. A relation to the test item was excluded as control animals were only
treated with the vehicle. Slight salivation was also observed for females during gestation and lactation. One female of test group 2 (300 mg/kg bw/d) showed a swelling at the body between PND 14 to 15. A swelling at the head (mouth region) was observed for female animal of test group 1 (100 mg/kg bw/d)
on PND 16. The swelling turned into a palpable mass on PND 17 and 18. One female animal of the control group did not properly nurse its pups and lost the complete litter on PND 2. All findings were assessed to occur incidentally and without a relation to the test item.

Mortality:

no mortality observed

Description (incidence):

No animal died prematurely in the present study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test substance-related changes in mean body weights and mean body weight change values were observed for male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) when compared to the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was slightly decreased in both sexes of test group 3 (1000 mg/kg bw/d) the first week of treatment. No additional changes were determined at later stages and no impact on body weight development was observed during the entire application period. Thus, these changes were potentially related to treatment but assessed to be without toxicological relevance. No deviations to control values were observed for male and female animals of test groups 1 and 2 (100 and 300 mg/
kg bw/d).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, non-treatment-related

Description (incidence and severity):

Water consumption was significantly increased in both sexes of test group 3 (1000 mg/kg bw/d) on premating days 7 and 14 (up to +30% in males and +24% in females). The same was true for these female animals on gestation day 14 (+37%) and on lactation day 10 (+61%). The finding was assessed to be induced by a bad taste of the test substance or local affection of the upper digestive tract and without toxicological relevance.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males of test groups 1 and 3 (100 and 1000 mg/kg bw/d) prothrombin time (Hepatoquick’s test, HQT) was shortened. The values were within the historical control range (HQT 33.1-40.9 sec) and, therefore, the changes were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males of test group 3 (1000 mg/kg bw/d) cholesterol values were increased and in females of the same test group total protein values were decreased. The values were slightly beyond historical control ranges (males: cholesterol 1.39-2.27 mmol/L; females: total protein 58.94-68.12 g/L). However, in each sex only one liver parameter was changed. Therefore, these changes were regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002). In females of test group 2 (300 mg/kg bw/d), total bile acid levels were lower when compared to controls, but the values were not dose-dependently changed. Therefore, this alteration was regarded as incidental and not treatment related.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

During detailed clinical observations (DCO), additional findings which did not already occur during the daily clinical examinations were not observed. One male and 2 female animals still showed salivation, a palpable mass through the skin was observed in each one female animal of test groups 0 (control) and 1 (100 mg/kg bw/d).
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, without a dose-response relationship or occurred in single animals only, these observations were considered as incidental.
Regarding the overall motor activity, no test substance-related deviations were noted for male and female animals.
Comparing the single intervals with the control groups, significantly decreased values were measured for male animals of test groups 2 and 3 (300 and 1000 mg/kg bw/d) at interval 1. The changes were regarded to be incidental and not related to treatment as neither other single intervals nor the overall motor activity was affected.
No deviations to control values were observed for female animals in test groups 1 to 3 (100, 300 and 1000 mg/kg bw/d) when compared to the control group.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The slight (grade 2) hypertrophy/hyperplasia of follicular cells of the thyroid glands in test group 3 females was considered as treatment-related.
The slightly increased incidence of altered colloid in test group 3 females and the slightly increased severity of altered colloid in test groups 2 and 3 females was assumed as treatment-related.
Both findings were assumed as treatment-related but non-adverse, since there were no correlating weight changes and the T4 levels in treated animals were within the normal range and did not indicate a functional impairment of thyroid glands in these animals.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Fertility
Female animal No. 127, which was not pregnant, as well as the male mating partner (No. 27) did not show relevant histopathological findings of reproductive organs. All other organs of these animals were not investigated histopathologically.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration in the different test groups (0-3) was between 3.93 days and 3.97 days.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The stages of spermatogenesis in the testes of males of test group 3 (1000 mg/kg bw/d) were comparable to those of the controls.

Reproductive performance:

no effects observed

Description (incidence and severity):

The male mating index calculated after the mating period for F1 litter was 100% in all test groups. The male fertility index was 80% in test group 0, 100% in test groups 1 and 3 and 90% in test group 2. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The female fertility index was 100% in test groups 1 and 3, 90% in test group 2 and 80% in test group 0. These values reflected the normal range of biological variation inherent in the strain of rats.
The gestation index was 100% in all test groups. The rate live birth indices were 100% in all test groups.
The postimplantation loss was 13.1% in test group 0 (control), 7.6% in test group 1 (100 mg/kg bw/d), 10.8% in test group 2 (300 mg/kg bw/d) and 7.3% in test group 3 (1000 mg/kg bw/d). These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data

Key result

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity, reproductive performance and fertility

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One male pup of test group 0 (control group) showed reduced nutritional condition (no milk in stomach) on lactation day 1. Two pups of test group 2 (300 mg/kg bw/d) died ahead of schedule. These findings were assessed to be spontaneous in nature. All other F1 pups in all test groups (0-3) did not show adverse clinical signs up to scheduled sacrifice on PND 4 and PND 13.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The viability index indicating pup mortality between PND 0 and 4 was 86.6% in test group 0 (control), 99.2% in test group 1 (100 mg/kg bw/d), 98.9% in test group 2 (300 mg/kg bw/d) and 99.1% in test group 3 (1000 mg/kg bw/d).The rate of liveborn pups in all test groups was not affected by the test substance, as indicated by live birth indices of 100.0% in test groups 0, 1 and 3 and 97.9% in test group 2.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group with the following exceptions.
Mean body weight of male pups in test group 1 (100 mg/kg bw/d) was significantly lower on lactation days 1 and 7 (-9.8%) when compared to control. In test group 3 (1000 mg/kg bw/d), body weight in male and female pups was significantly lower on lactation day 13 (-8.7% for males and -9.5% in females). In addition, the body weight change values of male and female pups were also lower on lactation days 7 and 13. However, as no clear dose-response relationship occurred and all values were covered by historical control values, the deviations to the control were assessed to be incidental and not related to the test item.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One male and one female pup of test group 3 (1000 mg/kg bw/d) showed a pale discolored liver. These findings were assessed to be incidental and not related to the test substance.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
Anogenital distance of male pups was significantly higher in test group 2 (300 mg/kg bw/d), the anogenital distance indices of male pups in test groups 1 and 2 (100 and 300 mg/kg bw/d) were also increased. In female animals of test group 2, anogenital distance and anogenital distance index showed increased values. However, as no dose-response relationship occurred, these changes were considered to be incidental and not treatment-related.
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.

No changes of T4 levels in males and females of the F0 generation as well as in male and female pups of the F1 generation at day 13 postpartum were observed.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 1: Male fertility indices for F0 males

	Test group 0 (0 mg/kg bw/d)	Test group 1 (100 mg/kg bw/d)	Test group 2 (300 mg/kg bw/d)	Test group 3 (1000 mg/kg bw/d)
Male fertility index [%]	80.0	100.0	90.0	100.0

*p<0.05;**p<0.01

The male mating index calculated after the mating period for F1 litter was 100% in all test groups.

Table 2: Fertility indices for F0 females

	Test group 0 (0 mg/kg bw/d)	Test group 1 (100 mg/kg bw/d)	Test group 2 (300 mg/kg bw/d)	Test group 3 (1000 mg/kg bw/d)
Female fertility index [%]	80	100	90	100

*p<0.05;**p<0.01

The female mating index calculated after the mating period for F1 litter was 100% in all test groups.

Table 3: Sex ratio of live F1 pups

PND 0	Test group 0 (0 mg/kg bw/d)	Test group 1 (100 mg/kg bw/d)	Test group 2 (300 mg/kg bw/d)	Test group 3 (1000 mg/kg bw/d)
Live males [%]	46.1	44.2	42.0	54.2
Live females [%]	53.9	55.8	58.0	45.8
PND 13
Live males [%]	53.6	50.0	43.1	50.0
Live females [%]	46.4	50.0	56.9	50.0

 

Table 4: Histopathology thyroid glands

	Female animals
Test group (mg/kg bw/d)	0 (0)	1 (100)	2 (300)	3 (1000)
No. of animals	10	10	10	10
Hypertrophy/hyperplasia, follicular	0	0	0	3
•  Grade 2				3
Altered colloid	6	6	6	8
•  Grade 1	6	6	4	5
•  Grade 2			2	3

 

Gestation index: The gestation index was 100% in all test groups.

Live birth indices: The rate live birth indices were 100% in all test groups.

Postimplantation loss: The postimplantation loss was 13.1% in test group 0 (control), 7.6% in test group 1 (100 mg/kg bw/d), 10.8% in test group 2 (300 mg/kg bw/d) and 7.3% in test group 3 (1000 mg/kg bw/d). These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Conclusions:

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of Aliquat 175 to Wistar rats revealed no adverse signs of toxicity up to a dose level of 1000 mg/kg bw/d in animals of both sexes.
Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d for male and female Wistar rats.
The NOAEL for reproductive performance and fertility was also set to 1000 mg/kg bw/d for male and female Wistar rats.
The NOAEL for developmental toxicity was 1000 mg/kg bw/d.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Studies performed according to OECD/EC guidelines and GLP principles (Klimisch 1).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A sub-acute oral study with screening for developmental/ reproduction effects was performed according to OECD/EC guidelines and GLP principles with MTBAC. MTBAC was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 31 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41-56 days). Formulation analysis confirmed that formulations were accurately and homogenously prepared. The parental NOAEL was determined to be 1000 mg/ kg bw/ day, based on absence of adverse effects at 1000 mg/ kg bw/ day. One couple in control group and one couple in highest dose group did not reproduce (female in highest dose group died on day 1 of meeting, death not substance related). No spermatogenic abnormalities were found. Gestation index and duration of gestation were not affected by treatment. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed. The number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related effects.

Based on these data, the parental, reproduction and developmental No Observed Adverse Effect Levels (NOAEL) for MTBAC were found to be >= 1000 mg/kg bw/ day.

A second study according OECD TG 422 and in compliance with GLP became available through a co-registrant. MTBAC was given daily as an aqueous solution to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 0, 100, 300 and 1000 mg/kg bw/day. Control animals were dosed daily with the vehicle water only. The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

Regarding clinical examinations, signs of general systemic toxicity were not observed in male or female parental animals of all test groups during the entire study period. Slight salivation shortly after test-substance administration was observed in all male and all female animals of the 1000 mg/kg bw/day test group as well as in 7 male and 7 female animals of the 300 mg/kg bw/day test group. This finding was not considered to be an adverse and toxicologically relevant effect.

Fertility indices for male and female animals were not impaired by test-substance administration even at a dose level of 1000 mg/kg bw/d. In addition, live birth indices of pups in all test groups were not influenced. The viability index as indicator for pup mortality was not altered. Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose level of the compound of 1000 mg/kg bw/day.

Regarding pathology, the thyroid gland in female animals was identified as the target organ. Female animals of 1000 mg/kg bw/day test group showed a slight hypertrophy/hyperplasia of follicular epithelial cells of the thyroid glands. Additionally, there was a slight increase in incidence and severity of altered colloid in the follicles in 300 and 1000 mg/kg bw/day test group females. Both findings were assumed as treatment-related but non-adverse, since there were no correlating weight changes and the T4 levels in treated animals were within the normal range and did not indicate a functional impairment of thyroid glands in these animals.

The NOAEL for general systemic toxicity was 1000 mg/kg bw/day for male and female Wistar rats. The NOAEL for reproductive performance and fertility was also set to 1000 mg/kg bw/day. The NOAEL for developmental toxicity was 1000 mg/kg bw/day.

Short description of key information:
Two oral OECD 422 screening studies performed with MTBAC, the parental and reproduction NOAEL was derived to be 1000 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
Two studies available with MTBAC.

Effects on developmental toxicity

Description of key information

 Two oral OECD 422 screening studies were performed with MTBAC (Klimisch 1 studies). The parental and developmental NOAEL was determined to be >=1000 mg/kg bw/day in both studies.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Remarks:

screening study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 May 2015 - 28 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: Organisation of Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (March 1996)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (July 2000)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 1995)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: See other Guidelines under "Principles of method if other than guideline"

Deviations:

no

Principles of method if other than guideline:

In addition, the procedures in this study essentially conformed to the following guidelines:

Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008);
OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008):
The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 10-12 weeks
- Weight at study initiation: 295-331 g (males); 207-231 g (females)
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages;
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages;
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages;
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany); All males and the selected 5 females/group were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy
- Water: Free access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 18 – 24 (temporary deviations from the daily mean relative humidity occurred, but laboratory historical data do not indicate an effect of the deviations)
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 May 2015 to 28 August 2015

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Method of formulation: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance and vehicle. No correction was made for the purity/composition of the test substance.

Storage conditions of formulations: At room temperature protected from light.

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dose preparations were taken at the test facility on a single occasion during the treatment period (formulations were prepared and sampled on 06 July 2015). The samples were dispatched on dry ice to second site where they were analyzed to assess accuracy of preparation (all groups), homogeneity (lowest and highest concentration) and stability in vehicle over 5 hours at room temperature protected from light (lowest and highest concentration).

No test substance was detected in the control group formulations. The concentrations analysed in the formulations of the test substance groups were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%). The formulations of the low and high dose groups prepared were homogeneous (i.e. coefficient of variation ≤ 10%).
Formulations at the entire range were stable when stored at room temperature protected from light for at least 6 hours (i.e. relative difference ≤ 10%).
The long term storage samples were stable at ≤-70°C for 7 days.

Details on mating procedure:

- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).

Duration of treatment / exposure:

Males were exposed for 31 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (actual: until day 5-7). Pups were not treated directly, but were potentially exposed to the test substance in utero, through lactational transfer or via exposure to maternal urine and/or faeces.

Frequency of treatment:

Once daily for 7 d/w.

Duration of test:

Males: 31 days
Females: 41-56 days

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:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were based on results of a 10-Day dose range finding study, in which 3 females per group were exposed to 500 or 1000 mg/ kg bw..
No mortality occurred in this study. At 500 mg/ kg bw/ day, all 3 rats showed salivation on 2-4 days. At 1000 mg/kg bw/ day, hunched posture was seen on 1-3 days for all 3 animals, flat gait on one day for 3 animals and salivation on 6-7 days for 3 animals. Furthermore, one animal showed piloerection on one day. Body weight gain was normal at 500 mg/ kg bw/ day, however at 1000 mg/ kg bw/ day 2 animals lost weight from day 1-5 and had slight weight gain from day 5-10. No effects were seen in either groups on food consumption and at macroscopic evaluation. Liver and kidney weights were normal for all treated animals.
Selection of animals for selected measurements (main study):
5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology. Only females with live offspring were selected. Histopathology was done on rats that died spontaneously (if possible).

Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.

Identification of pups:
On day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.

Maternal examinations:

CAGE SIDE OBSERVATIONS:
Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
Yes
- Time schedule: At least once daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals. This
was conducted after dosing at no specific time point, but within a similar time period after dosing for the respective animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT:
Yes
- Time schedule for examinations: 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 and 4.

FOOD CONSUMPTION:
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY:
Yes (Relative food consumption (g/kg body weight/day was calculated)

WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Yes
- Time schedule for examinations: The selected males were tested during week 4 of treatment and the selected females were tested during lactation (from lactation day 4 onwards).
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and locomotor activity (total movements and ambulations)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

SACRIFICE
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
No.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 1; many-toone 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 (Ref. 2; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 3) was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test (Ref. 4) was applied to motor activity data to determine intergroup differences.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Ref. 1 Dunnett C.W., A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
Ref. 2 Miller R.G., Simultaneous Statistical Inference, Springer Verlag, New York (1981).
Ref. 4 Fisher R.A., Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
Ref. 5 Kruskal W.H. and Wallis W.A.. Use of ranks in one-criterion variance analysis. Journal of the American Statistical Association 47 (260): 583-621, December (1952).

Indices:

For each group, the following calculations were performed:
- Mating index (%): (Number of females mated/Number of females paired) x 100
- Fertility index(%): (Number of pregnant females/Number of females paired) x 100
- Conception index(%): (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 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 of lactation: (Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check) x 100
- Viability index (%): (Number of live pups on day 4 of lactation / Number of pups born alive) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs of toxicity were noted during the observation period. Salivation seen after dosing among some animals of the 300 mg/kg bw/ day group and all animals of the 1000 mg/kg bw/ day dose group during was considered to be a physiological response 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). Alopecia was noted in one control female and was not considered related to the treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female at 1000 mg/kg bw/ day was found dead on day 1 of the mating period and showed beginning autolysis at necropsy (cause of death unclear). No other mortality occurred.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No treatment-related changes in body weights and body weight gain were noted in any of the groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment-related changes in food consumption before or after allowance for body weight were noted up to and including 1000 mg/kg bw/ day.

Food efficiency:

no effects observed

Description (incidence and severity):

No treatment-related changes in food consumption before or after allowance for body weight were noted up to and including 1000 mg/kg bw/ day.

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Description (incidence and severity):

No toxicologically relevant changes occurred in haematological parameters of treated rats. Incidental findings (increase in lymphocytes in males at 300 mg/kg bw/ day; increased platelet concentration in females at 100 mg/kg bw/ day) were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Several blood parameters were altered in males in the highest dose group (statistically significant): decreased total protein and albumin concentration, increased creatinine, and slightly increased potassium, chloride and inorganic phosphate levels. Reduced concentration of bile acids was observed in exposed male rats (reduction was statistically significant for mid and high dose group). It is of note that two control males were present with high bile acid concentration. As the values of exposed rats were still within the normal range, this effect was not considered toxicologically relevant. In parellel to males, also in the high dose females decreased total protein and albumin concentration was found (both statistically significant). Furthermore, statistically significant increase of aspartate aminotransferase (ASAT) activity was seen in this group. The level was however within the range considered normal for rats of this age and strain and the effect on ASAT was not considered to be toxicologically relevant. No other parameters were affected by the treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period. It was noted that the mean value for ambulations was lower in females at 1000 mg/kg bw/day, although not statistically significant. The difference was due to low values in 3/5 females and variation was high, also in the control group. Therefore, this difference was considered not to be related to treatment with the test substance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Absolute testes weights (average) were reduced in all three exposure groups (appr. -9.5%, -6.61% and -7.14% for the low, mid and high dose groups, respectively, compared to controls), but relative values were not affected. Together with the absence of a dose-relationship of the effect, this finding is found not to be toxicologically relevant. At 1000 mg/ kg bw, a reduced average spleen weight was seen in males (absolute and relative; reduction appr. 17% (statistically significant for relative weights only)). In females, absolute and relative thymus weight was decreased at 1000 mg/ kg bw/ day (appr. -14.5%), however this effect was not statistically significant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. Incidental observations included one male with dilated pelvics in the highest dose group. As this also was observed in 2 control males, this was not found to be related to MTBAC exposure. Furthermore, one animal had discoulered thymus at 1000 mg/kg bw/ day and 2 rats had nodules in their stomach (total number of affected animals = 2). At 100 mg/ kg bw/ day, one male was found with nodules in the epididymides, as this was only a single animal in the lowest dose group, this was not regarded as substance related effect. No gross macroscopic observations were reported for males exposed to 300 mg/kg bw/ day. In females, no gross pathological findings were reported for rats doset at 300 and 1000 mg/kg bw/ day. In the low dose group, one rat was found with focus/foci on its clitoral gland, this was judged to be an incidental finding and not related to MTBAC exposure. The female that was found dead during the study had discoloured kidneys, thymus with focus/ foci and discolouration of mesenteric lymph nodes. As the corpse was already in the process of autolysis, these observations are most likely not a direct effect of MTBAC exposure.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic findings. Minimal squamous cell hyperplasia of the forestomach, slight glandular erosion and slight hemorrhage and congestion of the glandular stomach (both correlated to several dark red foci of the glandular mucosa noted at necropsy) in the stomach of a single male of the 1000 mg/kg/day group. These findings were regarded to be caused by trauma during the gavage procedure and not directly related to the test item. Remaining recorded histologic changes were considered to be incidental findings. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Number of abortions:

no effects observed

Description (incidence and severity):

Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

One pup at 300 and 1000 mg/kg bw/day each were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Gestation index and duration of gestation were not affected by treatment.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Gestation index and duration of gestation were not affected by treatment. No signs of difficult or prolonged parturition were noted among the pregnant females.

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed at highest dose tested.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were considered to have been unaffected by treatment.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

One pup at 300 and 1000 mg/kg each were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was unaffected by treatment.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

Postnatal loss and viability index were unaffected by treatment.

External malformations:

no effects observed

Description (incidence and severity):

No abnormalities were noted in surviving pups. The pup that died at 300 mg/kg bw/day showed beginning autolysis and had no milk in the stomach. The pup that died at 1000 mg/kg bw/day had no abnormalities.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at 1000 mg/kg bw/day

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In an oral OECD 422 screening study with rats, the parental and developmental NOAEL was determined to be 1000 mg/kg bw/day.

Executive summary:

A sub-acute oral study with screening for developmental/ reproduction effects was performed according to OECD/EC guidelines and GLP principles with MTBAC. MTBAC was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 31 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41-56 days). Formulation analysis confirmed that formulations were accurately and homogenously prepared. The parental NOAEL was determined to be 1000 mg/ kg bw/ day, based on absence of adverse effects at 1000 mg/ kg bw/ day. One couple in the control group and one couple in the highest dose group did not reproduce (female in highest dose group died on day 1 of mating, death not substance related). No spermatogenic abnormalities were found. Gestation index and duration of gestation were not affected by treatment. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed. The number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related effects.

Based on these data, the parental and developmental No Observed Adverse Effect Levels (NOAEL) for MTBAC were found to be 1000 mg/kg bw/ day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Study performed according to OECD/EC guidelines and GLP principles (Klimisch 1).

Additional information

See discussion above on effects on reproduction.

Justification for classification or non-classification

Based on the available data, MTBAC is not classified for effects on reproduction and/ or development according to CLP Regulation (EC) No. 1272/2008.