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Developmental toxicity / teratogenicity

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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
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
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:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
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:
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:
other: OECD 421, Reproduction/Developmental Toxicity Screening Test (July 1995)
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
Deviations:
no
Qualifier:
according to
Guideline:
other: other guidelines as listed under "Principles of method if other than guideline"
Principles of method if other than guideline:
In addition, the procedures essentially conform 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
Specific details on test material used for the study:
No correction factor applied for purity/ composition.
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals 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 oral 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).
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.
Frequency of treatment:
Once daily, 7 d/w
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 (in the 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).
Positive control:
No
Observations and examinations performed and frequency:
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)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

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.

- Selected 5 animals/sex/group and all animals that died spontaneously or were killed in extremis.

- All remaining females which failed to deliver and the remaining males: According to test guidelines.

ORGAN WEIGHTS
- Selected 5 animals/sex/group (organs according to test guidelines).

- All remaining males:
Epididymides and testes

HISTOPATHOLOGY: Yes
According to test guidelines
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).
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 (in absence of clinical signs, it is unclear if this mortality was test substance-related). 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:
no effects observed
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):
no effects observed
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, 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 not statistically significant.
Gross pathological findings:
no effects observed
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
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
Dose descriptor:
NOAEL
Remarks:
Parental generation
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.
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
no

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 OECD422 screening study with MTBAC in rats, the NOAEL was determined to be 1000 mg/kg bw/day, based on no adverse effects seen at 1000 mg/kg bw/ day.
Executive summary:

A combined 28d repeated dose study with screening for reproductive and/ or developmental 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 bw/ day. 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. One female treated at 1000 mg/kg bw/ day was found dead on day 15 of the study, the death was found not to be treatment related. No further mortality occurred. No clinical signs were noted apart from salivation which was probably related to substance administration as it occurred in mid and high dose groups immediately after substance administration. Neurobehavioural examination did not show effects of MTBAC exposure. At 1000 mg/kg bw/ day, statistically significant decrease in total protein and albumin levels were noted in both sexes. In high dose males, in addition increased creatinine, potassium, chloride and inorganic phosphate concentrations were found. Spleen weights (absolute and relative) were decreased for males at 1000 mg/kg bw/ day (statistically significant). As no substance-related histopathological changes were seen and based on the fact that the observations had no apparent correlation, furthermore taking into consideration the low number of rats/ group (limiting statistical significance), these effects were not considered to be adverse. Therefore, a No Observed Adverse Effect Level (NOAEL) for MTBAC of ≥ 1000 mg/kg bw/ day was established.

Reason / purpose:
reference to same study
Reference
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:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
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:
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:
other: OECD 421, Reproduction/Developmental Toxicity Screening Test (July 1995)
Deviations:
no
Qualifier:
according to
Guideline:
other: See other Guidelines under "Principles of method if other than guideline"
Deviations:
no
Qualifier:
according to
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 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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
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:
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:
other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 1995)
Deviations:
no
Qualifier:
according to
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:
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Appearance: White powder with lumps and flakes
Test substance storage: At room temperature protected from light

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals 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

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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

Effect levels (maternal animals)

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.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

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

Effect levels (fetuses)

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

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

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.