Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

For this endpoint, an OECD 421 study in rats is available (Bentz, 2013). Three groups of ten male and ten female Sprague-Dawley rats received the test item daily by oral (gavage) from before mating, through mating and, for the females, through gestation until day 5 p.p. The test item was administered as an emulsion in the vehicle, drinking water treated by reverse osmosis, at dose-levels of 10, 30 or 100 mg/kg/day. Another group of ten males and ten females received the vehicle alone under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg/day was used. The animals were checked at least twice daily during the dosing period for mortality and morbidity and once daily for clinical signs. Body weight and food consumption were recorded at least once a week for males until sacrifice and at least once a week for females until mated. Body weight and food consumption of females were recorded on days 0, 7, 14 and 20 p.c. and days 1 and 5 p.p. The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5 p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. The pups were observed daily for clinical signs, abnormal behaviour and external abnormalities, and weighed on days 1 and 5 p.p. The males were sacrificed after 5 weeks of treatment and the dams on day 6p. p. Final body weights and selected organs weights (epididymides, liver, testes, thyroid with parathyroids, adrenals, brain, heart, kidneys, spleen, thymus) were recorded and a macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on selected organs from the control and high-dose groups (epididymides, liver, testes, ovaries, kidneys, stomach with forestomach) and the low- and mid-dose groups (forestomach of both sexes and kidneys of males), and on all macroscopic lesions. The pups were sacrificed on day 5 p.p. and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs.

There were no test item-related unscheduled deaths. Ptyalism was observed in males and females at 100 mg/kg/day throughout the whole study. There were no toxicologically relevant effects on male and female mean body weight, mean body weight gain or mean food consumption and on mean mating, fertility and delivery data. At pathology in males and females given 100 mg/kg/day, marked squamous cell hyperplasia with hyperkeratosis, occasionally associated with erosion/ulcer and inflammation was observed in the forestomach. These changes were considered to be adverse and correlated with gross observations (thickening and/or white or brown discoloration). In kidneys of males, increased incidence and severity of hyaline droplets was seen in proximal tubular cells. Immunohistological staining with an alpha-2µ-globulin antibody was performed in the kidneys from control and high-dose males. The positive staining confirmed that the eosinophilic hyaline droplets corresponded to alpha-2µ-globulin. As this alpha-2µ-globulin is specific to the rat, these higher incidence and severity seen in test item-treated males at 100 mg/kg/day were considered to be not relevant to human. The kidney changes probably correlated with the punctiform white discoloration seen at necropsy. There were no histological correlates to the slightly higher mean liver weight in females at 100 mg/kg/day (up to +12% from controls, p<0.05). At 30 mg/kg/day, minimal to slight squamous cell hyperplasia along with hyperkeratosis was observed associated with minimal inflammation (one male and one female) in the forestomach. In the absence of erosion/ulcer, changes at this dose-level were considered not to be adverse. No test item-related changes were seen in kidneys of males. At 10 mg/kg/day, no test item-related changes were seen either in the forestomach or in kidneys of males.

In pups, there were no test item-related deaths, clinical signs or necropsy findings and no toxicologically relevant effects on mean pup body weights, mean pup body weight gains or on the percentage of male pups at birth.

Based on the experimental conditions of this study:

- the NOEL for parental toxicity was considered to be 30 mg/kg/day based on forestomach adverse effects at the highest dose-level both in males and in females,

- the NOAEL for parental toxicity was considered to be 100 mg/kg/day in males and in females based on the lack of relevant systemic toxicity (excluding the local effects on the forestomach)

- the NOAEL for reproductive performance (mating and fertility) was considered to be 100 mg/kg/day,

- the NOAEL for toxic effects on progeny was considered to be 100 mg/kg/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 October 2012 - 15 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories Italia, Calco, Italy.
- Age at study initiation: the males were approximately 10 weeks old and the females were approximately 9 weeks on the day of treatment.
- Mean body weight at study initiation: the males had a mean body weight of 377 g (range: 349 g to 406 g) and the females had a mean body weight of 203 g (range: 177 g to 235 g) on the day of treatment.
- Fasting period before study: no
- Housing: the animals were individually housed, except during pairing and lactation, in polycarbonate cages (Tecniplast 2154, 940 cm²) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen since it is preferable for pregnant animals.
Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were provided to females and their litter as nesting material. Rat hut was given as environmental enrichment. The cages were placed in numerical order on the racks.
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 7 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 06 November 2012 to 31 December 2012
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The quantities of test item used for preparation of the dose formulations were corrected by a correction factor of 1.14 to take into account the water content and to express the doses in dry substance (taking into account tert-Amyl hydroperoxide and its impurities).
The test item was administered as an emulsion in the vehicle. The test item was mixed with the required quantity of vehicle.
The test item dose formulations were prepared with a frequency depending on their stability, stored at +2-8°C protected from light "prior-to-use" and delivered to the study room at room temperature and protected from light.

VEHICLE
- Choice of vehicle: the vehicle was drinking water treated by reverse osmosis.
- Concentration in vehicle: 0, 2, 6, and 20 mg/mL/day

Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): until mating occurred
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred to as day 0 post-coitum
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: HPLC-UV
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Homogeneity: assessed in homogeneity study: homogenous.
Stability: assessed in stability study: stable after 9 days at +4°C protected from light.
Duration of treatment / exposure:
In the males:
- 2 weeks before pairing,
- during the pairing period,
- until sacrifice (at least 5 weeks in total),

In the females:
- 2 weeks before pairing,
- during the pairing period,
- during gestation,
- during lactation until day 5 p.p. inclusive,
- until sacrifice for females with no delivery.
Frequency of treatment:
Daily
Details on study schedule:
- No F1 parents (only one generation mated)
- Age at mating of the mated animals in the study: approximately 11/12 weeks
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Remarks:
as t-amyl hydroperoxide
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
as t-amyl hydroperoxide
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
as t-amyl hydroperoxide
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected following the results of a previous non-GLP oral 2-week toxicity study. In this study, groups of three male and three female Sprague-Dawley rats were given the test item daily at doses of 40, 100 or 250 mg/kg/day as an emulsion in the same vehicle.
The dose-level of 250 mg/kg/day was considered to be too high to be chosen for the high dose level in the present study because of body weight loss, markedly reduced food consumption, several clinical signs such as piloerection, dyspnea, hunched posture and hypoactivity from the second week of treatment and local toxic effects on the forestomach (thickened and/or granular), stomach (white and/or black discolorations) and esophagus (thickened, perforation leading to the death of two animals which was considered to be related to the fragility of the esophagus wall following test item treatment).
At 100 mg/kg/day, the same local effects on the forestomach were seen at necropsy but the animals were in good clinical condition until the end of the study. The only clinical sign was ptyalism. There were no clear effects on body weight gains and mean food consumption were comparable to controls, except a minimal trend in females towards a reduction. Liver and kidneys weights were comparable to controls.
At 40 mg/kg/day, there were no effects on clinical signs, body weights, food consumption or organ weights. One male only had a granular forestomach at necropsy.
- Animal assignment: computerized stratification procedure
Positive control:
no (not required)
Parental animals: Observations and examinations:
MORTALITY/MORBIDITY:
- Time schedule: at least twice a day during the treatment period.

CLINICAL OBSERVATIONS:
- Time schedule: once a day during the treatment period.

BODY WEIGHT:
- Time schedule: Males: on the first day of treatment, then once a week until sacrifice. Females: on the first day of treatment, then once a week until mating, on days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

FOOD CONSUMPTION:
- Time schedule: once a week until sacrifice, but no recording during the mating period.

REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded
Oestrous cyclicity (parental animals):
Fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until females are mated.
Sperm parameters (parental animals):
Parameters examined in males of parental generation:
- testis weight (all groups) + microscopic evaluation (control and high-dose groups)
- epididymis weight (all groups) + microscopic evaluation (control and high-dose groups)
- special emphasis on stages of the spermatogenic cycle and testicular interstitial cells (control and high-dose groups)
Litter observations:
STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs

GROSS EXAMINATION OF DEAD AND SURVIVING PUPS:
- external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all animals after the end of the mating period.
- Female animals: all surviving animals = day 6 post-partum or, for females which had not delivered yet, day 25 post-coitum.

GROSS NECROPSY
A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all animals including any that was sacrificed prematurely.

HISTOPATHOLOGY
A microscopic examination was performed on:
- all tissues listed in the Table 1 from all animals of the control and high-dose groups (groups 1 and 4) sacrificed at the end of the treatment period (at the end of the pairing period for males or on day 6 p.p. for females) and from the females sacrificed for no delivery on day 25 p.c.,
- forestomach (both sexes) and kidneys (males) from animals of the low- and intermediate dose groups (groups 2 and 3) sacrificed at the end of the treatment period (at the end of the pairing period for males or on day 6 p.p. for females),
- all macroscopic lesions of all groups.



ORGAN WEIGHTS: see Table 1
Postmortem examinations (offspring):
SACRIFICE: on day 5 post-partum

GROSS NECROPSY: on all pups (surviving and found dead)

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No
Statistics:
Body weight, food consumption and reproductive data: Data are compared by one-way analysis of variances and Dunnett test (mean values being
considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions).
Organ weight: PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01) according to the sequenced described in the attached document.
Reproductive indices:
Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live pups) / Number of implantations
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)
Offspring viability indices:
Live birth index = 100 * (Number of live born pups / Number of delivered pups)
Viability index on day 4 p.p. = 100 * (Number of surviving pups on day 4 p.p. / Number of live born pups)
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
MORTALITY
One female given 100 mg/kg/day was prematurely sacrificed on day 2 p.p. following the death of its unique pup. This premature death was isolated and considered to be incidental.
At necropsy, no macroscopic changes were seen. At the histopathological examination, there was a moderate hyperplasia of squamous cells in the forestomach along with moderate hyperkeratosis. These observations were considered to be test item-related.
No test item-related changes were seen in other organs (kidneys, liver, ovaries/oviducts, stomach).

CLINICAL SIGNS
Ptyalism was observed in males and females at 100 mg/kg/day throughout the whole study. While test item-treatment related this finding was considered to be of minor toxicological importance.

BODY WEIGHT AND BODY WEIGHT CHANGE
There were no toxicologically important effects on male and female mean body weight and mean body weight gain.
The slight differences from controls seen in some mean body weight gains were considered not to be toxicologically relevant as they were without relevant impact on mean body weight, not dose related and/or not statistically significant.
The statistical level reached at 10 mg/kg/day in males for the interval days 15-22 was considered to be incidental.

FOOD CONSUMPTION
There were no effects on mean food consumptions

REPRODUCTIVE PERFORMANCE
Pairing, mating and fertility data

There were no test item-related effects on mating and fertility data.
All pairs mated within a maximum 4-day cohabitation period, with the exception of one pair at 100 mg/kg/day which mated in 2 weeks (the female was blocked in metestrous/diestrous for several days). This finding was isolated and considered to be incidental.

Delivery data
There were no toxicologically relevant effects on delivery data.

The slightly higher mean pre- and post-implantation losses at 100 mg/kg/day were particularly due to the above mentioned female (prematurely sacrificed for the death of its unique pup) with only a few corpora lutea and implantation sites (pre-implantation loss: 66.7%, post implantation loss: 50%). The slightly higher mean pre-implantation loss at 30 mg/kg/day was particularly due to one female whose pre-implantation loss (ten implants) were considered to be normal in view of the high number of corpora lutea (27).
Moreover mean pre- and post-implantation losses were within the ranges of our Historical Control Data.

ORGAN WEIGHTS
The mean absolute and relative liver weights were minimally but statistically significantly higher in females given 100 mg/kg/day (p<0.05) suggesting a test item-related effect. A similar, marginal and not statistically significant trend was also noted at this dose-level in males.

Other changes in mean organ weights were considered to be without relationship with the test item and part of the normal variation between groups.
The principal differences (expressed in %) noted between test item-treated and control animals in absolute and relative liver weights are summarized in the Table 2.

GROSS PATHOLOGY
Test item-related changes were seen at 30 mg/kg/day in the forestomach of one male and at 100 mg/kg/day in the forestomach of both sexes and in kidneys of males.

In the forestomach, thickening, white or brown discoloration were seen (see Table 3).

In kidneys, punctiform white discoloration was seen in 5/10 males given 100 mg/kg/day.

Other changes were considered to be part of the normal background commonly seen in rats.

HISTOPATHOLOGY
Test item-related changes were seen in the forestomach in both sexes at 30 and 100 mg/kg/day and in kidneys of males at 100 mg/kg/day.
. Forestomach
Hyperplasia of squamous cell was observed in the forestomach of both sexes at 30 and 100 mg/kg/day. This was graded minimal to slight at 30 mg/kg/day and moderate to marked at 100 mg/kg/day, correlating with macroscopic changes.
At 100 mg/kg/day, additional changes included hyperkeratosis and minimal to slight inflammation in most animals and erosion/ulcer in two males and one female. At 30 mg/kg/day, hyperkeratosis was present and minimal inflammation in one male and one female but no erosion/ulcer were observed.
Due to the high severity of changes in the forestomach at 100 mg/kg/day and the presence of erosion/ulcer, changes at this dose-level were considered to be adverse.
No test item-related changes were observed in the forestomach at 10 mg/kg/day.
The incidence and severity of these changes are summarized in the Table 4.

. Kidneys (males only)
Hyaline droplets were seen at a slightly higher incidence and severity in tubular epithelium of males given the test item at 100 mg/kg/day than in the other groups and may correlated with the punctiform white discoloration seen at necropsy.
Tubular basophilia was seen at a slightly higher incidence in treated groups than in controls but the distribution over the groups and the fact this was mainly observed unilaterally suggested that any relationship with the test item was unlikely.
The incidence and severity of these changes are summarized in the Table 5.
Immunohistological staining with an alpha 2 µ globulin antibody was performed in the kidneys from groups 1 (control) and 4 (high-dose) males.
A positive staining was observed in the tubular epithelium of kidneys in 6/10 animals from the control group and in 9/10 animals given 100 mg/kg/day. This was graded minimal or slight in controls and slight to moderate at 100 mg/kg/day.

This positive staining confirms that the eosinophilic hyaline droplets indeed corresponded to alpha 2 µ globulin, a protein commonly seen in male rats, and also confirms the higher incidence and severity in males given 100 mg/kg/day observed with the hematoxylin-eosin staining compared to controls.

As this alpha 2 µ globulin is specific to the rat, these higher incidence and severity seen in test item-treated males at 100 mg/kg/day were considered to be not relevant to human.

Other kidney changes were considered to be part of the normal background commonly seen in rats.

There were no histopathological changes in the liver which correlated with the slightly higher weight at necropsy.

Minimal focal subcapsular necrosis was seen in two females given 100 mg/kg/day. As this observation may be occasionally seen in liver of rats and as these foci were isolated and minimal, any relationship with the test item was considered to be unlikely.

Careful examination of testes and epidymides in males and ovaries in females did not show any test item-related changes.
Key result
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: forestomach adverse effects at the highest dose-level both in males and in females
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: for the lack of relevant systemic toxicity excluding the local effects on the stomach
Key result
Critical effects observed:
yes
System:
gastrointestinal tract
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
MORTALITY/VIABILITY/CLINICAL SIGNS:
Pup mortality and clinical signs are presented in the Table 8.

There were no test item-related deaths or clinical signs in pups.

BODY WEIGHT (GAIN):
There were no toxicologically relevant effects on mean pup body weights and mean pup body weight gains.

The statistical level reached at 30 mg/kg/day in male pups on day 1 p.p. was considered to be incidental.

GROSS PATHOLOGY:
There were no test item-related macroscopic findings at pup necropsy.

SEX RATIO:
There was no test item-related effect on the percentage of male pups at birth.


Key result
Dose descriptor:
NOAEL
Remarks:
toxic effects on progeny
Generation:
F1
Effect level:
100 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Reproductive effects observed:
no

Table 2  

Sex

Male

Female

Group

2

3

4

2

3

4

Dose-level (mg/kg/day)

10

30

100

10

30

100

Number of animals

10

10

10

10

8

9

Body weight

-1

+1

-1

+1

+1

+3

- Liver

 

 

 

 

 

 

  . absolute

+2

+4

+8

+2

+3

+12*

  . relative

+3

+4

+9

+1

+1

+9*

Statistically significant: *: p<0.05.

The significance concerned the organ weight values and not the percentages.

 

Table 3  

Sex

Male

Female

Group

1

2

3

4

1

2

3

4

Dose-level (mg/kg/day)

0

10

30

100

0

10

30

100

Number of animals

10

10

10

10

10

10

10

9

Thickening

-

-

1

9

-

-

-

3

White discoloration

-

-

-

8

-

-

-

-

Brown discoloration

-

-

-

1

-

-

-

-

- not observed in this group.

Table 4 

Sex

Male

Female

Group

1

2

3

4

1

2

3

4

Dose-level (mg/kg/day)

0

10

30

100

0

10

30

100

Number of animals

10

10

10

10

10

10

10

9

Hyperplasia; squamous cells

Minimal

-

-

1

-

-

-

6

-

Slight

-

-

4

-

-

-

2

-

Moderate

-

-

-

-

-

-

-

3

Marked

-

-

-

10

-

-

-

6

Total

0

0

5

10

0

0

8

9

Hyperkeratosis

Minimal

-

-

3

-

-

-

4

-

Slight

-

-

3

-

-

-

2

-

Moderate

-

-

-

-

-

-

-

3

Marked

-

-

-

10

-

-

-

6

Total

0

0

6

10

0

0

6

9

Erosion/ulcer

Minimal

-

-

-

1

-

-

-

-

Slight

-

-

-

1

-

-

-

1

Total

0

0

0

2

0

0

0

1

Inflammation

Minimal

-

-

1

6

-

-

1

5

Slight

-

-

-

4

-

-

-

1

Total

0

0

1

10

0

0

1

6

-: not observed in this group.

 

Table 5 

Sex

Male

Group

1

2

3

4

Dose-level (mg/kg/day)

0

10

30

100

Number of animals

10

10

10

10

Hyalin droplets in tubular epithelium

Minimal

4

4

5

3

Slight

-

-

-

6

Total

4

4

5

9

Tubular basophilia

Minimal

4

6

2

5

Slight

-

-

1

2

Total

4

6

3

7

-: not observed in this group.

 

Incidence and severity of hyaline droplets in kidney of controls and high-dose group males rats with hematoxylin/eosin and alpha 2 µ globulin stainings

 

Sex

Male

Group

1

4

1

4

Dose-level (mg/kg/day)

0

100

0

100

Number of animals

10

10

10

10

Staining

Hematoxylin-eosin

Alpha 2 µ globulin antibody

Hyalin droplets in tubular epithelium

Minimal

4

3

5

-

Slight

-

6

1

4

Moderate

-

-

-

5

Total

4

9

6

9

Conclusions:
The test item was administered daily by oral gavage to male and female Sprague Dawley rats, for 2 weeks before mating, during mating, gestation and until day 5 p.p., at dose-levels of 10, 30 or 100 mg/kg/day. Based on the experimental conditions of this study: the NOAEL for parental toxicity was considered to be 30 mg/kg/day based on forestomach adverse effects at the highest dose-level both in males and in females; the NOAEL for reproductive performance (mating and fertility) was considered to be 100 mg/kg/day, the NOAEL for toxic effects on progeny was considered to be 100 mg/kg/day.
Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until day 5 post-partum(p.p.), based on the OECD guideline No. 421, 27 July 1995. This study provides information on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

Three groups of ten male and ten female Sprague-Dawley rats received the test item daily by oral (gavage) from before mating, through mating and, for the females, through gestation until day 5p.p.. The test item was administered as an emulsion in the vehicle, drinking water treated by reverse osmosis, at dose-levels of 10, 30 or 100 mg/kg/day. Another group of ten males and ten females received the vehicle alone under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg/day was used. The animals were checked at least twice daily during the dosing period for mortality and morbidity and once daily for clinical signs. Body weight and food consumption were recorded at least once a week for males until sacrifice and at least once a week for females until mated. Body weight and food consumption of females were recorded on days 0, 7, 14 and 20p.c.and days 1 and 5p.p.. The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5p.p.. The total litter sizes and numbers of pups of each sex were recorded after birth. The pups were observed daily for clinical signs, abnormal behaviour and external abnormalities, and weighed on days 1 and 5p.p.. The males were sacrificed after 5 weeks of treatment and the dams on day 6p.p.. Final body weights and selected organs weights (epididymides, liver, testes, thyroid with parathyroids, adrenals, brain, heart, kidneys, spleen, thymus) were recorded and a macroscopicpost-mortemexamination of the principal thoracic and abdominal organs was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on selected organs from the control and high-dose groups (epididymides, liver, testes, ovaries, kidneys, stomach with forestomach) and the low- and mid-dose groups (forestomach of both sexes and kidneys of males), and on all macroscopic lesions. The pups were sacrificed on day 5p.p.and submitted for a macroscopicpost-mortemexamination of the principal thoracic and abdominal organs.

There were no test item-related unscheduled deaths.Ptyalism was observed in males and females at 100 mg/kg/day throughout the whole study.There were no toxicologically relevant effects on male and female mean body weight, mean body weight gain or mean food consumption and on mean mating, fertility and delivery data.At pathology in males and females given 100 mg/kg/day, marked squamous cell hyperplasia with hyperkeratosis, occasionally associated with erosion/ulcer and inflammation was observed in the forestomach. These changes were considered to be adverse and correlated with gross observations (thickening and/or white or brown discoloration). In kidneys of males, increased incidence and severity of hyaline droplets was seen in proximal tubular cells. Immunohistological staining with an alpha-2µ-globulin antibody was performed in the kidneys from control and high-dose males. The positive staining confirmed that the eosinophilic hyaline droplets corresponded to alpha-2µ-globulin.As this alpha-2µ-globulin is specific to the rat, these higher incidence and severity seen in test item-treated males at 100 mg/kg/day were considered to be not relevant to human. The kidney changes probably correlated with the punctiform white discoloration seen at necropsy. There were no histological correlates to the slightly higher mean liver weight in females at 100 mg/kg/day (up to +12% from controls, p<0.05). At 30 mg/kg/day, minimal to slight squamous cell hyperplasia along with hyperkeratosis was observed associated with minimal inflammation (one male and one female) in the forestomach. In the absence of erosion/ulcer, changes at this dose-level were considered not to be adverse. No test item-related changes were seen in kidneys of males. At 10 mg/kg/day, no test item-related changes were seen either in the forestomach or in kidneys of males.

In pups, there were no test item-related deaths, clinical signs or necropsy findings and no toxicologically relevanteffects on mean pup body weights, mean pup body weight gains or on the percentage of male pups at birth.

Based on the experimental conditions of this study:

- the NOEL for parental toxicity was considered to be 30 mg/kg/day based on forestomach adverse effects at the highest dose-level both in males and in females,

- the NOAEL for parental toxicity was considered to be 100 mg/kg/day in males and in females based on the lack of relevant systemic toxicity (excluding the local effects on the forestomach)

-  the NOAEL for reproductive performance (mating and fertility) was considered to be 100 mg/kg/day,

- the NOAEL for toxic effects on progeny was considered to be 100 mg/kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP guideline study.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to EU regulation N0. 1272/2008 (CLP), TAHP is not classified for reproductive toxicity.