Reaction Products of Diphosphorus Pentaoxide with Alcohols, C14-18 even, salted with Amines, C12-14, Tert-alkyl

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 September 2014 to 10 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

One female not paired on same day and target value of relative humidity was exceeded. (See below)

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Deviations:

yes

Remarks:

One female not paired on same day and target value of relative humidity was exceeded. (See below)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K Ltd., Blackthorn, Bicester, Oxon, UK
- Age at study initiation: (P) 12 weeks
- Weight at study initiation: (P) Males: 287-342 g; Females: 186-219 g
- Fasting period before study: overnight
- Housing: solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. Transfer to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis during pairing. If evidence of successful mating, males were returned to their original cages. Mated females were housed individually in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes during gestation and lactation
- Diet (e.g. ad libitum): Rodent 2018C Teklad Global Certified Pelleted Diet, Harlan Laboratories U.K Ltd., Oxon, UK
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3 °C
- Humidity (%): 50+/-20 %
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 hours continuous light/dark

IN-LIFE DATES: From: 25 September 2014 To: 20 November 2014

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

Arachis oil BP

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was diluted in Arachis oil BP

VEHICLE
- Justification for use and choice of vehicle (if other than water): Arachis oil was used as the substance formed stable homogenous solutions in this vehicle
- Concentration in vehicle: Low – 100 mg/ml
Intermediate – 300 mg/ml
High – 750 mg/ml ((reduced to 500 mg/kg bw/day from Day 17 onwards due to two deaths and adverse clinical signs at 750 mg/kg bw/day). Following the reduction at the high dose level the time weighted average at this level was calculated to be 595 mg/kg bw/day (based on an average of 42 days of treatment).

- Amount of vehicle (if gavage): 4mL/kg

Details on mating procedure:

- M/F ratio per cage: animals paired on a 1 male:1 female basis within each dose group at Day 15. One female was removed from pairing on Day 16 due to clinical condition and then re-paired with her male partner on Day 19.
- Length of cohabitation: maximum of 14 days
- Proof of pregnancy: Presence of sperm in vaginal smear and/or vaginal plug in situ referred to as day 0of gestation.
- No replacement of first male by another male with proven fertility if unsuccessful mating.
- After successful mating each pregnant female was caged (how): After evidence of mating (designated as Day 0 post coitum), the males were returned to their original cages. Mated females were transferred to individual cages during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the substance in the test material formulations was determined by chemical analysis and all formulations were within +/- 9% of nominal.

Duration of treatment / exposure:

Test duration:
- 42 days for males
- Until 5 days post partum for females
- Recovery group males and females were maintained without treatment for a further 14 days after sacrifice of the non-recovery males

Frequency of treatment:

The substance was administered daily, for 42 consecutive days, by gavage (except for females during parturition where applicable).

Details on study schedule:

Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum.

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

750 mg/kg bw/day reduced to 500 mg/kg bw/day from Day 17 onwards due to two deaths and adverse clinical signs at 750 mg/kg bw/day.

No. of animals per sex per dose:

12 animals of each sex (m/f) were allocated to each dose group. In addition, 5 animals of each sex (m/f) were allocated to each control goup. 12 males 13 females for high dose group and 5 M 4 F for recovery group at 750/500 due to switching of one female of the recovery group to the non-recovery group at high dose.

Control animals:

yes, concurrent vehicle

other: recovery control (concurrent vehicle)... (see attached file)

Details on study design:

- Dose selection rationale: Range finding study and 28 days
- Rationale for animal assignment (if not random): Random
- Other: Recovery group animals were maintained for a further fourteen days treatment-free period following termination of treatment
- Control animals were treated in an identical manner with 4 ml/kg/day of Arachis oil BP
- The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

Positive control:

Not used

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, soon after dosing and 1 hour after dosing (except for females during parturition where applicable). Recovery animals observed once daily during the treatment-free period.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 and then weekly for males until termination and weekly for females until pairing. During pairing, daily for females until mating was confirmed. (Days 0, 7, 14 and 20 post coitum and Days 1 and 4 post partum for females). Day 1 and weekly thereafter for recovery group animals.

FOOD CONSUMPTION:
Food consumption was recorded during the pre-pairing period for each cage group at weekly intervals throughout the study. For males after the mating phase. For females showing evidence of mating during the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters during the lactation period (Days 1-4)

WATER CONSUMPTION:
Water intake was observed daily by visual inspection of water bottles for any overt changes.

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Not examined

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number of offspring born, number of offspring alive recorded daily and reported on Days 1 and 4 post partum, sex of offspring on Days 1 and 4 post partum, clinical condition of offspring from birth to Day 5 post partum, weight, surface righting reflex on Day 1 post partum.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal examination and any macroscopic abnormalities; possible cause of death was examined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals at Day 43
- Maternal animals: All surviving animals at Day 5 post partum for females that have offspring and at Day 25 post coitum or after for females without offspring

GROSS NECROPSY
Full external and internal examination, any macroscopic abnormalities were recorded

HISTOPATHOLOGY / ORGAN WEIGHTS
- Histopathology: Yes Samples from the following tissues were removed from all animals and preserved in 10% buffered formalin except where stated
- Organ weight:
The following organs were weighted: epididymides and testes
The following tissues were examined: Coagulating gland, Epididymes, Ovaries, Mammary gland (females only), Pituitary, Prostate, Seminal vesicles, Testes, Uterus/Cervix, Vagina

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 5 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

Statistics:

Data were processed to give summary incidence or group mean and standard deviation values where appropriate. All data were summarised in tabular form.
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters: Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed below:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:

p<0.01
p<0.05
p≥0.05 (not significant)

Reproductive indices:

Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
- Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
- Fertility Indices
For each group the following were calculated:
Mating Index (%) = Number of animals paired/Number of animals mated x 100
Pregnancy Index (%) = Number of animals mated/Number of pregnant females x 100

Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
- Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
- Parturition Index
The following was calculated for each group:
Parturition Index (%) = Number of pregnant females/Number of females delivering live offspring x 100

Offspring viability indices:

The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
- Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
Pre–implantation loss (%) =Number of corpora lutea - Number of implantation sites/Number of corpora lutea x100
Post–implantation loss (%) = Number of implantation sites - Total number of offspring born/Number of implantation sites x100
- Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = Number of offspring alive on Day 1/Number of offspring born x 100
Viability Index (%) = Number of offspring alive on Day 4/Number of offspring alive on Day 1 x100
- Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the
following formula: Number of male offspring/ Total number of offspring x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

2 females found dead on Day 9 and 11 at 750 mg/kg bw/day. See details on results for clinical signs.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduction in body weight gain during week 2 and 3 for non-recovery males and a statistically significant reduction during week 1 and 3 in recovery males at 750 mg/kg bw/day. Reduction during the final week of gestation for females at 750 mg/kg bw/day

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduction in body weight gain during week 2 and 3 for non-recovery males and a statistically significant reduction during week 1 and 3 in recovery males at 750 mg/kg bw/day. Reduction during the final week of gestation for females at 750 mg/kg bw/day

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

1 control female, 1 female treated with 100 mg/kg bw/day, 1 female at 750 mg/kg bw/day were not pregnant following positive evidence of mating. No histopathological evidence in reproductive organs of both sexes for these non fertile pairings.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- Mortality: Two females treated with 750/500 mg/kg bw/day were found dead on Day 9 and 11. There were no further unscheduled deaths.
- Clinical signs: Episodes of increased salivation were evident in the majority of treated animals throughout the treatment period. The onset and incidence showed a dose related response to treatment. Instances of wet and stained fur were evident in a number of females treated with 750/500 mg/kg bw/day. One female from this treatment group had tiptoe gait, hunched posture, body tremors, stained ano-genital region, noisy/laboured respiration and a decreased respiratory rate on Day 16. A further female from this treatment group also had body tremors on Day 16. Following the reduction of the high dose level the incidence and severity of the clinical signs (excluding increased salivation) subsided.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Body weight: Non-recovery males treated with 750/500 mg/kg bw/day showed a reduction in body weight gain during Weeks 2 and 3. Recovery males treated with 750/500 mg/kg bw/day showed a statistically significant reduction in body weight gain during Weeks 1 and 3. Recovery in all males was evident thereafter. Females from this treatment group showed a reduction in body weight on Day 20 of gestation and in body weight gain during the final week of gestation. No such effects were detected in animals of either sex treated with 300 or 100 mg/kg bw/day.
- Food consumption: No treatment-related effects on dietary intake were noted for males during the study or for females during the pre-pairing, gestation or lactation phases of the study. Females treated with 100 mg/kg bw/day showed a statistically significant increase in food consumption during the first week of gestation. An increase in food consumption is not considered to represent an adverse effect of treatment therefore the intergroup difference was considered not to be of toxicological importance. Fluctuations in food conversion efficiency were evident in males treated with 750/500 mg/kg bw/day however these followed the reductions in body weight gain and therefore were considered not to represent an adverse effect of treatment.
- Water consumption: No treatment-related effect on water consumption was evident in treated animals when compared to controls.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Mating: No treatment-related effects were detected in mating performance. One female treated with 750/500 mg/kg bw/day did not show any signs of mating and was not pregnant.
- Fertility: There were no treatment-related differences in fertility. One control female, one female treated with 100 mg/kg bw/day and one female treated with 750/500 mg/kg bw/day were not pregnant following positive evidence of mating. No histopathological correlates were evident in the female or male reproductive organs of any of these non fertile pairings which could have been the cause of the infertility.
- Gestation lenght: There were no differences in gestation lengths. The distribution for the majority of treated females was compared to controls. Gestation lengths were between 21½ and 23½ days. One female treated with 750/500 mg/kg bw/day that had a total litter loss had a gestation length of 25 Days. It is not unusual for females with such small litters to fail to maintain their litter especially with an extended gestation period however the remaining females that had a total litter loss showed normal gestation lengths.

ORGAN WEIGHTS (PARENTAL ANIMALS): No treatment-related changes were evident in the organ weights measured. Statistical analysis of the data did not reveal any significant intergroup differences.

GROSS PATHOLOGY (PARENTAL ANIMALS): The female that was found dead on Day 11 had a distended stomach. A thin non-glandular region with pale areas was also evident. These observations are indicative of toxicity and may have contributed to the death of this female. The female that was found dead on Day 9 had her ovaries, uterus, cervix and vagina cannibalised. No macroscopic abnormalities were detected in the remaining tissues therefore a cause of death could not be established. However in view of the second death and clinical signs at this high dose level a treatment related effect can not be excluded.
There were no macroscopic abnormalities detected in terminal kill animals.

HISTOPATHOLOGY (PARENTAL ANIMALS): There were no treatment-related microscopic abnormalities detected.

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Clinical signs:

no effects observed

Description (incidence and severity):

Only incidental clinical signs: small size, cold, weak, pale, no milk in stomach, physical injury but considered to be low incidence findings and unrelated to test item toxicity. See results below.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Five females had a total litter losses either at birth or by Day 1 post partum

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

In total eleven females from the control and 100 mg/kg bw/day dose groups, ten females from the 300 mg/kg bw/day dose group and seven females from the 750/500 mg/kg bw/day dose group gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

VIABILITY (OFFSPRING): Three females treated with 750/500 mg/kg bw/day and two females treated with 300 mg/kg bw/day had a total litter loss either at birth or by Day 1 post partum. No significant treatment-related effects were detected for corpora lutea, implantation counts, implantation losses, litter size or litter viability for treated animals when compared to controls. A slight reduction in the number of corpora lutea was evident in females treated with 750/500 mg/kg bw/day, subsequently a slight reduction in implantation sites and the number of offspring born was also evident. Statistical analysis of the data did not reveal any significant intergroup differences and the majority of individual values were within normal historical ranges for rats of the strain and age used. A direct effect on corpora lutea is also generally uncommon following a short treatment period prior to mating. Therefore the intergroup differences were considered most likely to represent normal biological variation. There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

CLINICAL SIGNS (OFFSPRING): No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, cold, weak, pale, no milk in stomach, physical injury, missing or found dead were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.

BODY WEIGHT (OFFSPRING): There were no toxicologically significant effects detected. Statistical analysis of the litter, offspring weights or surface righting reflex data did not reveal any significant intergroup differences.

SEXUAL MATURATION (OFFSPRING): Not examined

ORGAN WEIGHTS (OFFSPRING): Not examined

GROSS PATHOLOGY (OFFSPRING): No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

not specified

The oral administration of the test material to rats for a period of up to eight weeks (including two weeks pre-pairing, gestation and early lactation for females) at dose levels of 100, 300 and 750/500 mg/kg bw/day (time weighted average calculated to be 595 mg/kg bw/day (based on an average of 42 days of treatment)), resulted in treatment-related effects in animals of either sex treated with 750/500 mg/kg bw/day and females treated with 300 mg/kg bw/day. Clinical signs were detected in animals of either sex from all treatment groups during the study. Episodes of increased salivation were reported with the onset of increased salivation showing a dose related response to treatment. Prior to the reduction of the high dose level two females were found dead on Day 9 and 11. By Day 16, instances of wet and stained fur were evident in a number of high dose females. One female from this treatment group had tiptoe gait, hunched posture, body tremors, stained ano-genital region, noisy/laboured respiration and a decreased respiratory rate on Day 16. A further female from this treatment group also had body tremors on Day 16. Following the reduction of the high dose level these clinical signs subsided and the worst affected female successfully littered and maintained a live litter until Day 5 post partum. The other female that showed occasional tremors was not pregnant. The physical condition of males treated with 750/500 mg/kg bw/day was also slightly affected with reductions in body weight development during Weeks 1, 2 and 3 of treatment. Recovery was evident thereafter and therefore the reductions may be considered to represent an initial response to treatment. Females from this treatment group also showed a reduction in body weight gain during the final week of gestation. Mating and fertility performance was unaffected by treatment however three females treated with 750/500 mg/kg bw/day and two females treated with 300 mg/kg bw/day had a total litter loss either at birth or by Day 1 post partum. One female treated with 750/500 mg/kg bw/day was non-mated and a further female from this treatment group was not pregnant. For both of these parameters, the incidences were considered within normal limits for this type of study, however, it did mean a smaller number of litters to evaluate. For litters reared to Day 5 of age there was no obvious adverse effect on survival or development of the offspring. Within the confines of this screening study it is not possible to determine if the total litter losses were or were not related to maternal toxicity or frank reproductive effects. No such effects were evident at 100 mg/kg bw/day. Given the results noted in the 750/500 mg/kg bw/day group, the number of litter losses in the 300 mg/kg bw/day group cannot be discounted as a treatment effect. However, litter losses at the 300 mg/kg bw/day group were at the high range of controls or what can be regarded as normal biological variation. Further, the lack of effect on offspring litter size and body weight gain suggests that maternal toxicity may be the cause of the total litter losses rather than poor offspring viability.

Conclusions:

The oral administration of the test material to rats by gavage, at dose levels of 100, 300 and 750/500 mg/kg bw/day, resulted in treatment-related effects indicative of maternal toxicity in females treated with 750/500 mg/kg bw/day and equivocal results for reproductive toxicity at 750/500 and 300 mg/kg bw/day. Due to the equivocal nature of the test results, it is difficult to state with certainty that reproductive effects have been observed. Thus, 100 mg/kg bw/day is considered to be the NOEL for general toxicity potentially affecting reproduction.

Executive summary:

GUIDELINE

The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, and provides an initial hazard assessment and contributes to decisions with respect to the necessity and timing of additional testing (OECD 421, Section 5). The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995). This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

METHODS

The test item was administered by gavage to three groups, each of twelve male and twelve (thirteen for high dose group) female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 750 mg/kg bw/day (reduced to 500 mg/kg bw/day from Day 17 onwards due to two deaths and adverse clinical signs at 750 mg/kg bw/day). Following the reduction at the high dose level the time weighted average at this level was calculated to be 595 mg/kg bw/day (based on an average of 42 days of treatment). A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five (four for treated group) females, were treated with the high dose (750/500 mg/kg bw/day) or the vehicle alone (Arachis oil BP) for forty two consecutive days and then maintained without treatment for a further fourteen days. In view of the premature deaths and clinical signs at the high dosage, it was decided to prioritise the non-recovery group animals to try to ensure that there were sufficient litters at this dosage for meaningful assessment. One female from the recovery high dose group was therefore designated as a non-recovery animal. Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. Pairing of non-recovery animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 (excluding No 16 which was paired on Day 16 and No.s 85 and 112 which were paired on Day 19) of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Non-recovery adult males were terminated on Day 43, followed by the termination of all surviving non-recovery females and offspring on Day 5 post partum. Any female which failed to mate or which did not produce a pregnancy was terminated on or after Day 25 post coitum. Recovery animals were terminated on Day 57. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

RESULTS

Adult Responses

Mortality

Two females treated with 750/500 mg/kg bw/day were found dead on Day 9 and 11. The female that was found dead on Day 11 had a distended stomach with a thin non-glandular region and pale areas were also evident. These observations are indicative of toxicity and may have contributed to the death of this female. There were no further unscheduled deaths.

Clinical Observations

Episodes of increased salivation were evident in the majority of treated animals throughout the treatment period. Instances of wet and stained fur were evident in a number of females treated with 750/500 mg/kg bw/day. One female from this treatment group had incidences of tiptoe gait, hunched posture, body tremors, stained ano-genital region, noisy/laboured respiration and a decreased respiratory rate, despite this apparent toxicity, this female produced and reared a viable litter to Day 5 post partum. A further female from this treatment group also had an isolated incident of body tremors.

Body Weight

Non-recovery males treated with 750/500 mg/kg bw/day showed a reduction in body weight gain during Weeks 2 and 3. Recovery males treated with 750/500 mg/kg bw/day showed a statistically significant reduction in body weight gain during Weeks 1 and 3. Recovery of body weights in all males was evident thereafter. Females from this treatment group showed a reduction in body weight gain during the final week of gestation. No such effects were detected in animals of either sex treated with 300 or 100 mg/kg bw/day.

Food Consumption

No treatment-related effects were detected in food consumption or food conversion efficiency for treated males throughout the treatment period or for treated females during maturation, gestation or lactation.

Water Consumption

No treatment-related effect on water consumption was evident in treated animals when compared to controls. Reproductive Performance Mating No treatment-related effects were detected in mating performance.

Fertility

There were no treatment-related differences in fertility. Gestation Length There were no differences in gestation lengths. The distribution for the majority of treated females was comparable to controls. Gestation lengths were between 21½ and 23½ days. One female treated with 750/500 mg/kg bw/day that had a total litter loss had a gestation length of 25 Days.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

Three females treated with 750/500 mg/kg bw/day and two females treated with 300 mg/kg bw/day had a total litter loss either at birth or by day 1 post partum. Thus five out of forty four total litters were lost. Thirty nine total litters survived. Of the surviving litters born, no treatment related effects in litter size at birth and subsequently on Days 1 and 4 post partum were evident. Sex ratio in treated litters was comparable to controls.

Offspring Growth and Development

Offspring body weight gain and litter weights at birth and subsequently on Days 1 and 4 post partum were comparable to controls. Surface righting reflex in treated litters was comparable to controls.

Offspring Observations

The clinical signs and necropsy findings apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 100, 300 or 750/500 mg/kg bw/day.

Pathology

Necropsy

At 750/500 mg/kg bw/day the female that was found dead on Day 11 had a distended stomach with a thin non-glandular region and pale areas also evident. These observations are indicative of toxicity and may have contributed to the death of this female. The female that was found dead on Day 9 had her ovaries, uterus, cervix and vagina cannibalised. No macroscopic abnormalities were detected in the remaining tissues therefore a cause of death could not be established. However in view of the second death and clinical signs at this high dose level a treatment related effect can not be excluded. There were no macroscopic abnormalities detected in terminal kill animals.

Organ Weights

No treatment-related changes were evident in the organ weights measured.

Histopathology

There were no treatment-related microscopic abnormalities detected.

CONCLUSION

The oral administration of the test material to rats by gavage, at dose levels of 100, 300 and 750/500 mg/kg bw/day, resulted in treatment-related effects indicative of maternal toxicity in females treated with 750/500 mg/kg bw/day and equivocal results for reproductive toxicity at 750/500 and 300 mg/kg bw/day. Due to the equivocal nature of the test results, it is difficult to state with certainty that reproductive effects have been observed. Thus, 100 mg/kg bw/day is considered to be the NOEL for general toxicity potentially affecting reproduction.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

GUIDELINE

The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, and provides an initial hazard assessment and contributes to decisions with respect to the necessity and timing of additional testing (OECD 421, Section 5). The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995). This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

METHODS

The test item was administered by gavage to three groups, each of twelve male and twelve (thirteen for high dose group) female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 750 mg/kg bw/day (reduced to 500 mg/kg bw/day from Day 17 onwards due to two deaths and adverse clinical signs at 750 mg/kg bw/day). Following the reduction at the high dose level the time weighted average at this level was calculated to be 595 mg/kg bw/day (based on an average of 42 days of treatment). A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five (four for treated group) females, were treated with the high dose (750/500 mg/kg bw/day) or the vehicle alone (Arachis oil BP) for forty two consecutive days and then maintained without treatment for a further fourteen days. In view of the premature deaths and clinical signs at the high dosage, it was decided to prioritise the non-recovery group animals to try to ensure that there were sufficient litters at this dosage for meaningful assessment. One female from the recovery high dose group was therefore designated as a non-recovery animal. Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. Pairing of non-recovery animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 (excluding No 16 which was paired on Day 16 and No.s 85 and 112 which were paired on Day 19) of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Non-recovery adult males were terminated on Day 43, followed by the termination of all surviving non-recovery females and offspring on Day 5 post partum. Any female which failed to mate or which did not produce a pregnancy was terminated on or after Day 25 post coitum. Recovery animals were terminated on Day 57. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

RESULTS

Adult Responses

Mortality

Two females treated with 750/500 mg/kg bw/day were found dead on Day 9 and 11. The female that was found dead on Day 11 had a distended stomach with a thin non-glandular region and pale areas were also evident. These observations are indicative of toxicity and may have contributed to the death of this female. There were no further unscheduled deaths.

Clinical Observations

Episodes of increased salivation were evident in the majority of treated animals throughout the treatment period. Instances of wet and stained fur were evident in a number of females treated with 750/500 mg/kg bw/day. One female from this treatment group had incidences of tiptoe gait, hunched posture, body tremors, stained ano-genital region, noisy/laboured respiration and a decreased respiratory rate, despite this apparent toxicity, this female produced and reared a viable litter to Day 5 post partum. A further female from this treatment group also had an isolated incident of body tremors.

Body Weight

Non-recovery males treated with 750/500 mg/kg bw/day showed a reduction in body weight gain during Weeks 2 and 3. Recovery males treated with 750/500 mg/kg bw/day showed a statistically significant reduction in body weight gain during Weeks 1 and 3. Recovery of body weights in all males was evident thereafter. Females from this treatment group showed a reduction in body weight gain during the final week of gestation. No such effects were detected in animals of either sex treated with 300 or 100 mg/kg bw/day.

Food Consumption

No treatment-related effects were detected in food consumption or food conversion efficiency for treated males throughout the treatment period or for treated females during maturation, gestation or lactation.

Water Consumption

No treatment-related effect on water consumption was evident in treated animals when compared to controls. Reproductive Performance Mating No treatment-related effects were detected in mating performance.

Fertility

There were no treatment-related differences in fertility. Gestation Length There were no differences in gestation lengths. The distribution for the majority of treated females was comparable to controls. Gestation lengths were between 21½ and 23½ days. One female treated with 750/500 mg/kg bw/day that had a total litter loss had a gestation length of 25 Days.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

Three females treated with 750/500 mg/kg bw/day and two females treated with 300 mg/kg bw/day had a total litter loss either at birth or by day 1 post partum. Thus five out of forty four total litters were lost. Thirty nine total litters survived. Of the surviving litters born, no treatment related effects in litter size at birth and subsequently on Days 1 and 4 post partum were evident. Sex ratio in treated litters was comparable to controls.

Offspring Growth and Development

Offspring body weight gain and litter weights at birth and subsequently on Days 1 and 4 post partum were comparable to controls. Surface righting reflex in treated litters was comparable to controls.

Offspring Observations

The clinical signs and necropsy findings apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 100, 300 or 750/500 mg/kg bw/day.

Pathology

Necropsy

At 750/500 mg/kg bw/day the female that was found dead on Day 11 had a distended stomach with a thin non-glandular region and pale areas also evident. These observations are indicative of toxicity and may have contributed to the death of this female. The female that was found dead on Day 9 had her ovaries, uterus, cervix and vagina cannibalised. No macroscopic abnormalities were detected in the remaining tissues therefore a cause of death could not be established. However in view of the second death and clinical signs at this high dose level a treatment related effect can not be excluded. There were no macroscopic abnormalities detected in terminal kill animals.

Organ Weights

No treatment-related changes were evident in the organ weights measured.

Histopathology

There were no treatment-related microscopic abnormalities detected.

CONCLUSION

The oral administration of the test material to rats by gavage, at dose levels of 100, 300 and 750/500 mg/kg bw/day, resulted in treatment-related effects indicative of maternal toxicity in females treated with 750/500 mg/kg bw/day and equivocal results for reproductive toxicity at 750/500 and 300 mg/kg bw/day. Due to the equivocal nature of the test results, it is difficult to state with certainty that reproductive effects have been observed. Thus, 100 mg/kg bw/day is considered to be the NOEL for general toxicity potentially affecting reproduction.

Short description of key information:

Test item was administered by gavage to three groups, each of twelve male and twelve (thirteen for high dose group) female Wistar Han:RccHan:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 750 mg/kg bw/day (reduced to 500mg from Day 17 onwards due to two deaths and adverse clinical signs at 750 mg/kg bw/day).

The oral administration of the test material to rats by gavage, at dose levels of 100, 300 and 750/500 mg/kg bw/day, resulted in treatment-related effects indicative of maternal toxicity in females treated with 750/500 mg/kg bw/day and equivocal results for reproductive toxicity at 750/500 and 300 mg/kg bw/day. Due to the equivocal nature of the test results, it is difficult to state with certainty that reproductive effects have been observed. Thus, 100 mg/kg bw/day is considered to be the NOEL for general toxicity potentially affecting reproduction. No litter losses or adverse effect on offspring development was observed from maternal treatment at 100 mg/kg bw/day (OECD 421).

Effects on developmental toxicity

Description of key information

Test item was administered by gavage to three groups, each of twelve male and twelve (thirteen for high dose group) female Wistar Han:RccHan:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 750 mg/kg bw/day (reduced to 500mg from Day 17 onwards due to two deaths and adverse clinical signs at 750 mg/kg bw/day).

The oral administration of the test material to rats by gavage, at dose levels of 100, 300 and 750/500 mg/kg bw/day, resulted in treatment-related effects indicative of maternal toxicity in females treated with 750/500 mg/kg bw/day and equivocal results for reproductive toxicity at 750/500 and 300 mg/kg bw/day. Due to the equivocal nature of the test results, it is difficult to state with certainty that reproductive effects have been observed. Thus, 100 mg/kg bw/day is considered to be the NOEL for general toxicity potentially affecting reproduction. No litter losses or adverse effect on offspring development was observed from maternal treatment at 100 mg/kg bw/day (OECD 421).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 September 2014 to 10 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

One female not paired on same day and target value of relative humidity was exceeded. (See below)

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Deviations:

yes

Remarks:

One female not paired on same day and target value of relative humidity was exceeded. (See below)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K Ltd., Blackthorn, Bicester, Oxon, UK
- Age at study initiation: (P) 12 weeks
- Weight at study initiation: (P) Males: 287-342 g; Females: 186-219 g
- Fasting period before study: overnight
- Housing: solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. Transfer to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis during pairing. If evidence of successful mating, males were returned to their original cages. Mated females were housed individually in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes during gestation and lactation
- Diet (e.g. ad libitum): Rodent 2018C Teklad Global Certified Pelleted Diet, Harlan Laboratories U.K Ltd., Oxon, UK
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3 °C
- Humidity (%): 50+/-20 %
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 hours continuous light/dark

IN-LIFE DATES: From: 25 September 2014 To: 20 November 2014

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

Arachis oil BP

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was diluted in Arachis oil BP

VEHICLE
- Justification for use and choice of vehicle (if other than water): Arachis oil was used as the substance formed stable homogenous solutions in this vehicle
- Concentration in vehicle:
Low – 100 mg/ml
Intermediate – 300 mg/ml
High – 750 mg/ml ((reduced to 500 mg/kg bw/day from Day 17 onwards due to two deaths and adverse clinical signs at 750 mg/kg bw/day). Following the reduction at the high dose level the time weighted average at this level was calculated to be 595 mg/kg bw/day (based on an average of 42 days of treatment).

- Amount of vehicle (if gavage): 4mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the substance in the test material formulations was determined by chemical analysis and all formulations were within +/- 9% of nominal.

Details on mating procedure:

- M/F ratio per cage: animals paired on a 1 male:1 female basis within each dose group at Day 15. One female was removed from pairing on Day 16 due to clinical condition and then re-paired with her male partner on Day 19.
- Length of cohabitation: maximum of 14 days
- Proof of pregnancy: Presence of sperm in vaginal smear and/or vaginal plug in situ referred to as day 0of gestation.
- No replacement of first male by another male with proven fertility if unsuccessful mating.
- After successful mating each pregnant female was caged (how): After evidence of mating (designated as Day 0 post coitum), the males were returned to their original cages. Mated females were transferred to individual cages during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes

Duration of treatment / exposure:

Test duration:
- 42 days for males
- Until 5 days post partum for females
- Recovery group males and females were maintained without treatment for a further 14 days after sacrifice of the non-recovery males

Frequency of treatment:

The substance was administered daily, for 42 consecutive days, by gavage (except for females during parturition where applicable).

Duration of test:

Approximately 6 weeks.

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

750 mg/kg bw/day reduced to 500 mg/kg bw/day from Day 17 onwards due to two deaths and adverse clinical signs at 750 mg/kg bw/day.

No. of animals per sex per dose:

12 animals of each sex (m/f) were allocated to each dose group. In addition, 5 animals of each sex (m/f) were allocated to each control goup. 12 males 13 females for high dose group and 5 M 4 F for recovery group at 750/500 due to switching of one female of the recovery group to the non-recovery group at high dose.

Control animals:

yes, concurrent vehicle

other: recovery control (concurrent vehicle)... (see attached file)

Details on study design:

- Dose selection rationale: Range finding study and 28 days
- Rationale for animal assignment (if not random): Random
- Other: Recovery group animals were maintained for a further fourteen days treatment-free period following termination of treatment
Control animals were treated in an identical manner with 4 ml/kg/day of Arachis oil BP
The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals

Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, soon after dosing and 1 hour after dosing (except for females during parturition where applicable). Recovery animals observed once daily during the treatment-free period.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 and then weekly for males until termination and weekly for females until pairing. During pairing, daily for females until mating was confirmed. (Days 0, 7, 14 and 20 post coitum and Days 1 and 4 post partum for females). Day 1 and weekly thereafter for recovery group animals.

FOOD CONSUMPTION:
Food consumption was recorded during the pre-pairing period for each cage group at weekly intervals throughout the study. For males after the mating phase. For females showing evidence of mating during the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters during the lactation period (Days 1-4)

WATER CONSUMPTION:
Water intake was observed daily by visual inspection of water bottles for any overt changes.

SACRIFICE
- Male animals: All surviving animals at Day 43
- Maternal animals: All surviving animals at Day 5 post partum for females that have offspring and at Day 25 post coitum or after for females without offspring
- The offsprings were sacrificed at 5 days of age.

GROSS NECROPSY
Full external and internal examination, any macroscopic abnormalities were recorded for all animals and offspring, including those dying during the study.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Histopathology: Yes Samples from the following tissues were removed from all animals and preserved in 10% buffered formalin except where stated
- Organ weight:
The following organs was weighted: epididymides and testes
The following tissues was examined: Coagulating gland, Epididymes, Ovaries, Mammary gland (females only), Pituitary, Prostate, Seminal vesicles, Testes, Uterus/Cervix, Vagina

Ovaries and uterine content:

PREGNANCY AND PARTURITION
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends. The following was recorded for each female: Date of pairing,Date of mating, Date and time of observed start of parturition, Date and time of observed completion of parturition.

Fetal examinations:

LITTER DATA
PARAMETERS EXAMINED
The following parameters were examined for each litter:
[number of offspring born, number of offspring alive recorded daily and reported on Days 1 and 4 post partum, sex of offspring on Days 1 and 4 post partum, clinical condition of offspring from birth to Day 5 post partum, weight.

PHYSICAL DEVELOPMENT
- All alive offspring were assessed for surface righting reflex on Day 1 post partum.

Statistics:

Data were processed to give summary incidence or group mean and standard deviation values were appropriate. All data were summarised in tabular form.
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed below:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:

p<0.01
p<0.05
p≥0.05 (not significant)

Indices:

Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
- Pre-coital Interval calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
- Fertility Indices
For each group:
Mating Index (%) = Number of animals paired/Number of animals mated x 100
Pregnancy Index (%) = Number of animals mated/Number of pregnant females x 100

Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
- Gestation Length calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
- Parturition Index
For each group:
Parturition Index (%) = Number of pregnant females/Number of females delivering live offspring x 100
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
- Implantation Losses (%) Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
Pre–implantation loss (%) =Number of corpora lutea - Number of implantation sites/Number of corpora lutea x100
Post–implantation loss (%) = Number of implantation sites - Total number of offspring born/Number of implantation sites x100
- Live Birth and Viability Indices
For each litter:
Live Birth Index (%) = Number of offspring alive on Day 1/Number of offspring born x 100
Viability Index (%) = Number of offspring alive on Day 4/Number of offspring alive on Day 1 x100
- Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:
Number of male offspring/ Total number of offspring x 100

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- Mortality: Two females treated with 750/500 mg/kg bw/day were found dead on Day 9 and 11. There were no further unscheduled deaths.
- Clinical signs: Episodes of increased salivation were evident in the majority of treated animals throughout the treatment period. The onset and incidence showed a dose related response to treatment. Instances of wet and stained fur were evident in a number of females treated with 750/500 mg/kg bw/day. One female from this treatment group had tiptoe gait, hunched posture, body tremors, stained ano-genital region, noisy/laboured respiration and a decreased respiratory rate on Day 16. A further female from this treatment group also had body tremors on Day 16. Following the reduction of the high dose level the incidence and severity of the clinical signs (excluding increased salivation) subsided.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Body weight: Non-recovery males treated with 750/500 mg/kg bw/day showed a reduction in body weight gain during Weeks 2 and 3. Recovery males treated with 750/500 mg/kg bw/day showed a statistically significant reduction in body weight gain during Weeks 1 and 3. Recovery in all males was evident thereafter. Females from this treatment group showed a reduction in body weight on Day 20 of gestation and in body weight gain during the final week of gestation. No such effects were detected in animals of either sex treated with 300 or 100 mg/kg bw/day.
- Food consumption: No treatment-related effects on dietary intake were noted for males during the study or for females during the pre-pairing, gestation or lactation phases of the study. Females treated with 100 mg/kg bw/day showed a statistically significant increase in food consumption during the first week of gestation. An increase in food consumption is not considered to represent an adverse effect of treatment therefore the intergroup difference was considered not to be of toxicological importance. Fluctuations in food conversion efficiency were evident in males treated with 750/500 mg/kg bw/day however these followed the reductions in body weight gain and therefore were considered not to represent an adverse effect of treatment.
- Water consumption: No treatment-related effect on water consumption was evident in treated animals when compared to controls.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Mating: No treatment-related effects were detected in mating performance. One female treated with 750/500 mg/kg bw/day did not show any signs of mating and was not pregnant.
- Fertility: There were no treatment-related differences in fertility. One control female, one female treated with 100 mg/kg bw/day and one female treated with 750/500 mg/kg bw/day were not pregnant following positive evidence of mating. No histopathological correlates were evident in the female or male reproductive organs of any of these non fertile pairings which could have been the cause of the infertility.
- Gestation lenght: There were no differences in gestation lengths. The distribution for the majority of treated females was compared to controls. Gestation lengths were between 21½ and 23½ days. One female treated with 750/500 mg/kg bw/day that had a total litter loss had a gestation length of 25 Days. It is not unusual for females with such small litters to fail to maintain their litter especially with an extended gestation period however the remaining females that had a total litter loss showed normal gestation lengths.

ORGAN WEIGHTS (PARENTAL ANIMALS): No treatment-related changes were evident in the organ weights measured. Statistical analysis of the data did not reveal any significant intergroup differences.

GROSS PATHOLOGY (PARENTAL ANIMALS): The female that was found dead on Day 11 had a distended stomach. A thin non-glandular region with pale areas was also evident. These observations are indicative of toxicity and may have contributed to the death of this female. The female that was found dead on Day 9 had her ovaries, uterus, cervix and vagina cannibalised. No macroscopic abnormalities were detected in the remaining tissues therefore a cause of death could not be established. However in view of the second death and clinical signs at this high dose level a treatment related effect can not be excluded.
There were no macroscopic abnormalities detected in terminal kill animals.

HISTOPATHOLOGY (PARENTAL ANIMALS): There were no treatment-related microscopic abnormalities detected.


In total eleven females from the control and 100 mg/kg bw/day dose groups, ten females from the 300 mg/kg bw/day dose group and seven females from the 750/500 mg/kg bw/day dose group gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

VIABILITY (OFFSPRING): Three females treated with 750/500 mg/kg bw/day and two females treated with 300 mg/kg bw/day had a total litter loss either at birth or by Day 1 post partum. No significant treatment-related effects were detected for corpora lutea, implantation counts, implantation losses, litter size or litter viability for treated animals when compared to controls. A slight reduction in the number of corpora lutea was evident in females treated with 750/500 mg/kg bw/day, subsequently a slight reduction in implantation sites and the number of offspring born was also evident. Statistical analysis of the data did not reveal any significant intergroup differences and the majority of individual values were within normal historical ranges for rats of the strain and age used. A direct effect on corpora lutea is also generally uncommon following a short treatment period prior to mating. Therefore the intergroup differences were
considered most likely to represent normal biological variation. There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

CLINICAL SIGNS (OFFSPRING): No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, cold, weak, pale, no milk in stomach, physical injury, missing or found dead were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.

BODY WEIGHT (OFFSPRING): There were no toxicologically significant effects detected. Statistical analysis of the litter, offspring weights or surface righting reflex data did not reveal any significant intergroup differences.

SEXUAL MATURATION (OFFSPRING): Not examined

ORGAN WEIGHTS (OFFSPRING): Not examined

GROSS PATHOLOGY (OFFSPRING): No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

mortality

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

not specified

Relevant for humans:

not specified

The oral administration of the test material to rats for a period of up to eight weeks (including two weeks pre-pairing, gestation and early lactation for females) at dose levels of 100, 300 and 750/500 mg/kg bw/day (time weighted average calculated to be 595 mg/kg bw/day (based on an average of 42 days of treatment)), resulted in treatment-related effects in animals of either sex treated with 750/500 mg/kg bw/day and females treated with 300 mg/kg bw/day. Clinical signs were detected in animals of either sex from all treatment groups during the study. Episodes of increased salivation were reported with the onset of increased salivation showing a dose related response to treatment. Prior to the reduction of the high dose level two females were found dead on Day 9 and 11. By Day 16, instances of wet and stained fur were evident in a number of high dose females. One female from this treatment group had tiptoe gait, hunched posture, body tremors, stained ano-genital region, noisy/laboured respiration and a decreased respiratory rate on Day 16. A further female from this treatment group also had body tremors on Day 16. Following the reduction of the high dose level these clinical signs subsided and the worst affected female successfully littered and maintained a live litter until Day 5 post partum. The other female that showed occasional tremors was not pregnant. The physical condition of males treated with 750/500 mg/kg bw/day was also slightly affected with reductions in body weight development during Weeks 1, 2 and 3 of treatment. Recovery was evident thereafter and therefore the reductions may be considered to represent an initial response to treatment. Females from this treatment group also showed a reduction in body weight gain during the final week of gestation. Mating and fertility performance was unaffected by treatment however three females treated with 750/500 mg/kg bw/day and two females treated with 300 mg/kg bw/day had a total litter loss either at birth or by Day 1 post partum. One female treated with 750/500 mg/kg bw/day was non-mated and a further female from this treatment group was not pregnant. For both of these parameters, the incidences were considered within normal limits for this type of study, however, it did mean a smaller number of litters to evaluate. For litters reared to Day 5 of age there was no obvious adverse effect on survival or development of the offspring. Within the confines of this screening study it is not possible to determine if the total litter losses were or were not related to maternal toxicity or frank reproductive effects. No such effects were evident at 100 mg/kg bw/day. Given the results noted in the 750/500 mg/kg bw/day group, the number of litter losses in the 300 mg/kg bw/day group cannot be discounted as a treatment effect. However, litter losses at the 300 mg/kg bw/day group were at the high range of controls or what can be regarded as normal biological variation. Further, the lack of effect on offspring litter size and body weight gain suggests that maternal toxicity may be the cause of the total litter losses rather than poor offspring viability.

Conclusions:

The oral administration of the test material to rats by gavage, at dose levels of 100, 300 and 750/500 mg/kg bw/day, resulted in treatment-related effects indicative of maternal toxicity in femalestreated with 750/500 mg/kg bw/day and equivocal results for reproductive toxicity at 750/500 and 300 mg/kg bw/day.

Executive summary:

GUIDELINE

The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, and provides an initial hazard assessment and contributes to decisions with respect to the necessity and timing of additional testing (OECD 421, Section 5). The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995). This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

METHODS

The test item was administered by gavage to three groups, each of twelve male and twelve (thirteen for high dose group) female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 750 mg/kg bw/day (reduced to 500 mg/kg bw/day from Day 17 onwards due to two deaths and adverse clinical signs at 750 mg/kg bw/day). Following the reduction at the high dose level the time weighted average at this level was calculated to be 595 mg/kg bw/day (based on an average of 42 days of treatment). A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five (four for treated group) females, were treated with the high dose (750/500 mg/kg bw/day) or the vehicle alone (Arachis oil BP) for forty two consecutive days and then maintained without treatment for a further fourteen days. In view of the premature deaths and clinical signs at the high dosage, it was decided to prioritise the non-recovery group animals to try to ensure that there were sufficient litters at this dosage for meaningful assessment. One female from the recovery high dose group was therefore designated as a non-recovery animal. Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. Pairing of non-recovery animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 (excluding No 16 which was paired on Day 16 and No.s 85 and 112 which were paired on Day 19) of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Non-recovery adult males were terminated on Day 43, followed by the termination of all surviving non-recovery females and offspring on Day 5 post partum. Any female which failed to mate or which did not produce a pregnancy was terminated on or after Day 25 post coitum. Recovery animals were terminated on Day 57. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

RESULTS

Adult Responses

Mortality

Two females treated with 750/500 mg/kg bw/day were found dead on Day 9 and 11. The female that was found dead on Day 11 had a distended stomach with a thin non-glandular region and pale areas were also evident. These observations are indicative of toxicity and may have contributed to the death of this female. There were no further unscheduled deaths.

Clinical Observations

Episodes of increased salivation were evident in the majority of treated animals throughout the treatment period. Instances of wet and stained fur were evident in a number of females treated with 750/500 mg/kg bw/day. One female from this treatment group had incidences of tiptoe gait, hunched posture, body tremors, stained ano-genital region, noisy/laboured respiration and a decreased respiratory rate, despite this apparent toxicity, this female produced and reared a viable litter to Day 5 post partum. A further female from this treatment group also had an isolated incident of body tremors.

Body Weight

Non-recovery males treated with 750/500 mg/kg bw/day showed a reduction in body weight gain during Weeks 2 and 3. Recovery males treated with 750/500 mg/kg bw/day showed a statistically significant reduction in body weight gain during Weeks 1 and 3. Recovery of body weights in all males was evident thereafter. Females from this treatment group showed a reduction in body weight gain during the final week of gestation. No such effects were detected in animals of either sex treated with 300 or 100 mg/kg bw/day.

Food Consumption

No treatment-related effects were detected in food consumption or food conversion efficiency for treated males throughout the treatment period or for treated females during maturation, gestation or lactation.

Water Consumption

No treatment-related effect on water consumption was evident in treated animals when compared to controls. Reproductive Performance Mating No treatment-related effects were detected in mating performance.

Fertility

There were no treatment-related differences in fertility. Gestation Length There were no differences in gestation lengths. The distribution for the majority of treated females was comparable to controls. Gestation lengths were between 21½ and 23½ days. One female treated with 750/500 mg/kg bw/day that had a total litter loss had a gestation length of 25 Days.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

Three females treated with 750/500 mg/kg bw/day and two females treated with 300 mg/kg bw/day had a total litter loss either at birth or by day 1 post partum. Thus five out of forty four total litters were lost. Thirty nine total litters survived. Of the surviving litters born, no treatment related effects in litter size at birth and subsequently on Days 1 and 4 post partum were evident. Sex ratio in treated litters was comparable to controls.

Offspring Growth and Development

Offspring body weight gain and litter weights at birth and subsequently on Days 1 and 4 post partum were comparable to controls. Surface righting reflex in treated litters was comparable to controls.

Offspring Observations

The clinical signs and necropsy findings apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 100, 300 or 750/500 mg/kg bw/day.

Pathology

Necropsy

At 750/500 mg/kg bw/day the female that was found dead on Day 11 had a distended stomach with a thin non-glandular region and pale areas also evident. These observations are indicative of toxicity and may have contributed to the death of this female. The female that was found dead on Day 9 had her ovaries, uterus, cervix and vagina cannibalised. No macroscopic abnormalities were detected in the remaining tissues therefore a cause of death could not be established. However in view of the second death and clinical signs at this high dose level a treatment related effect can not be excluded. There were no macroscopic abnormalities detected in terminal kill animals.

Organ Weights

No treatment-related changes were evident in the organ weights measured.

Histopathology

There were no treatment-related microscopic abnormalities detected.

CONCLUSION

The oral administration of the test material to rats by gavage, at dose levels of 100, 300 and 750/500 mg/kg bw/day, resulted in treatment-related effects indicative of maternal toxicity in females treated with 750/500 mg/kg bw/day and equivocal results for reproductive toxicity at 750/500 and 300 mg/kg bw/day. Due to the equivocal nature of the test results, it is difficult to state with certainty that reproductive effects have been observed. Thus, 100 mg/kg bw/day is considered to be the NOEL for general toxicity potentially affecting reproduction.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

GUIDELINE

The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, and provides an initial hazard assessment and contributes to decisions with respect to the necessity and timing of additional testing (OECD 421, Section 5). The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995). This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

METHODS

The test item was administered by gavage to three groups, each of twelve male and twelve (thirteen for high dose group) female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 750 mg/kg bw/day (reduced to 500 mg/kg bw/day from Day 17 onwards due to two deaths and adverse clinical signs at 750 mg/kg bw/day). Following the reduction at the high dose level the time weighted average at this level was calculated to be 595 mg/kg bw/day (based on an average of 42 days of treatment). A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five (four for treated group) females, were treated with the high dose (750/500 mg/kg bw/day) or the vehicle alone (Arachis oil BP) for forty two consecutive days and then maintained without treatment for a further fourteen days. In view of the premature deaths and clinical signs at the high dosage, it was decided to prioritise the non-recovery group animals to try to ensure that there were sufficient litters at this dosage for meaningful assessment. One female from the recovery high dose group was therefore designated as a non-recovery animal. Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. Pairing of non-recovery animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 (excluding No 16 which was paired on Day 16 and No.s 85 and 112 which were paired on Day 19) of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Non-recovery adult males were terminated on Day 43, followed by the termination of all surviving non-recovery females and offspring on Day 5 post partum. Any female which failed to mate or which did not produce a pregnancy was terminated on or after Day 25 post coitum. Recovery animals were terminated on Day 57. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

RESULTS

Adult Responses

Mortality

Two females treated with 750/500 mg/kg bw/day were found dead on Day 9 and 11. The female that was found dead on Day 11 had a distended stomach with a thin non-glandular region and pale areas were also evident. These observations are indicative of toxicity and may have contributed to the death of this female. There were no further unscheduled deaths.

Clinical Observations

Episodes of increased salivation were evident in the majority of treated animals throughout the treatment period. Instances of wet and stained fur were evident in a number of females treated with 750/500 mg/kg bw/day. One female from this treatment group had incidences of tiptoe gait, hunched posture, body tremors, stained ano-genital region, noisy/laboured respiration and a decreased respiratory rate, despite this apparent toxicity, this female produced and reared a viable litter to Day 5 post partum. A further female from this treatment group also had an isolated incident of body tremors.

Body Weight

Non-recovery males treated with 750/500 mg/kg bw/day showed a reduction in body weight gain during Weeks 2 and 3. Recovery males treated with 750/500 mg/kg bw/day showed a statistically significant reduction in body weight gain during Weeks 1 and 3. Recovery of body weights in all males was evident thereafter. Females from this treatment group showed a reduction in body weight gain during the final week of gestation. No such effects were detected in animals of either sex treated with 300 or 100 mg/kg bw/day.

Food Consumption

No treatment-related effects were detected in food consumption or food conversion efficiency for treated males throughout the treatment period or for treated females during maturation, gestation or lactation.

Water Consumption

No treatment-related effect on water consumption was evident in treated animals when compared to controls. Reproductive Performance Mating No treatment-related effects were detected in mating performance.

Fertility

There were no treatment-related differences in fertility. Gestation Length There were no differences in gestation lengths. The distribution for the majority of treated females was comparable to controls. Gestation lengths were between 21½ and 23½ days. One female treated with 750/500 mg/kg bw/day that had a total litter loss had a gestation length of 25 Days.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

Three females treated with 750/500 mg/kg bw/day and two females treated with 300 mg/kg bw/day had a total litter loss either at birth or by day 1 post partum. Thus five out of forty four total litters were lost. Thirty nine total litters survived. Of the surviving litters born, no treatment related effects in litter size at birth and subsequently on Days 1 and 4 post partum were evident. Sex ratio in treated litters was comparable to controls.

Offspring Growth and Development

Offspring body weight gain and litter weights at birth and subsequently on Days 1 and 4 post partum were comparable to controls. Surface righting reflex in treated litters was comparable to controls.

Offspring Observations

The clinical signs and necropsy findings apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 100, 300 or 750/500 mg/kg bw/day.

Pathology

Necropsy

At 750/500 mg/kg bw/day the female that was found dead on Day 11 had a distended stomach with a thin non-glandular region and pale areas also evident. These observations are indicative of toxicity and may have contributed to the death of this female. The female that was found dead on Day 9 had her ovaries, uterus, cervix and vagina cannibalised. No macroscopic abnormalities were detected in the remaining tissues therefore a cause of death could not be established. However in view of the second death and clinical signs at this high dose level a treatment related effect can not be excluded. There were no macroscopic abnormalities detected in terminal kill animals.

Organ Weights

No treatment-related changes were evident in the organ weights measured.

Histopathology

There were no treatment-related microscopic abnormalities detected.

CONCLUSION

The oral administration of the test material to rats by gavage, at dose levels of 100, 300 and 750/500 mg/kg bw/day, resulted in treatment-related effects indicative of maternal toxicity in females treated with 750/500 mg/kg bw/day and equivocal results for reproductive toxicity at 750/500 and 300 mg/kg bw/day. Due to the equivocal nature of the test results, it is difficult to state with certainty that reproductive effects have been observed. Thus, 100 mg/kg bw/day is considered to be the NOEL for general toxicity potentially affecting reproduction.

Justification for classification or non-classification

Classification as a reproductive toxicant category 2 is not required because Regulation (EC) No. 1272/2008 (CLP) states that this decision should be based on effects observed in the absence of other toxic effects or, if occurring together with other toxic effects, the adverse effect on reproduction should be considered not to be a secondary non-specific consequence of the other toxic effects. Furthermore, the key 28-day repeated dose oral toxicity study reported a NOAEL of 750 mg/kg bw/day for males and females and stated that adverse effects seen in rats were not relevant to humans.