3,4-dimethylbenzaldehyde

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

SafePharm Laboratories Ltd., Shardlow Business Park, London Road, Shardlow, Derby, DE72 2GD, UK

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, Kent
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: males 250-305 g, females 169-210 g.
- Fasting period before study: No
- Housing: Initially, all animals were housed in groups of five in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper. During the mating phase, animals were transferred to similar cages on a one male:one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes. Environmental enrichment was provided in the form of wooden chew blocks (B&K Universal Ltd, Hull, UK) and cardboard fun tunnels (Datesand Ltd, Cheshire, UK) except for mated females during gestation and lactation.
- Diet: A pelleted diet (Rodent PMI 5002 (Certified) diet, BCM IPS Ltd., London, UK, ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ±2
- Humidity (%): 55±15
- Air changes (per hr): ≥15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was prepared at the appropriate concentrations as a solution in Polyethylene glycol 400. The stability and homogeneity of the test material formulations were determined. The formulations were stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4°C in the dark.

VEHICLE
- Amount of vehicle: 4 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually during the period of gestation and lactation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test material formulation were taken on a weekly basis and analysed for concentration of 3,4-Dimethylbenzaldehyde. The concentration of 3, 4-Dimethyl Benzaldehyde in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique. The test material formulations were diluted with acetonitrile to give a final, theoretical test material concentration of approximately 0.05 mg/mL. Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 0.05 mg/mL. For homogeneity determinations, the test material formulations were mixed thoroughly and. samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate. For stability determinations, the test material formulations were sampled and analysed initially and then after storage at approximately +4°C in the dark for fourteen days. The results indicate that the analysed formulations were within acceptable limits for the purpose of this study.

Duration of treatment / exposure:

up to forty-six consecutive days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
50, 250, 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: based on preliminary range-finder in the rat

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
- All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before and after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before and after dosing, and one hour after dosing at weekends (except for females during parturition where applicable).

BODY WEIGHT: Yes
- Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination. Females were weighed weekly until mating was evident. Bodyweights were then recorded on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, determined as g/animal/day
- During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded during the period of gestation. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Food efficiency (the ratio of bodyweight change/dietary intake) was calculated retrospectively for males throughout the study period and for females during maturation and the first two weeks of gestation. Due to offspring growth and milk production, food efficiency could not be accurately calculated during the final week of gestation and during lactation.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 14 (day prior to pairing) and at termination
- Animals fasted: No
- How many animals: 5 males and 5 females of each test and control group
- Parameters examined: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), Erythrocyte indices: [mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC)], Total leucocyte count (WBC), Differential leucocyte count: [neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas), Reticulocyte count (Retic; cresyl blue stained slides were prepared but reticulocytes were not assessed)], Platelet count (PLT), Prothrombin time (CT), Activated thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 14 (day prior to pairing) and at termination
- Animals fasted: No
- How many animals: 5 males and 5 females of each test and control group
- Parameters examined: Urea, Glucose, Total protein, Albumin, Albumin/Globulin (A/G) ratio (by calculation), Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium(Ca++), Inorganic phosphorus (P), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Alkaline phosphatase (AP), Creatinine, Total cholesterol, Total bilirubin.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females per dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
- Dose groups that were examined: all
- Battery of functions tested: behavioural assessment, functional performance tests (motor activity, Forelimb/Hindlimb Grips Strength), Sensory reactivity

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number of offspring born, number and sex of pups alive recorded daily and reported on Day 1 and 4 post partum, clinical condition, weight gain, reflexological response (surface righting reflex).

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals study day 43, following completion of the female gestation and lactation phases.
- Maternal animals: All surviving animals at day 5 post partum.

GROSS PATHOLOGY: Yes, a full external and internal examination, and any macroscopic abnormalities were recorded. In addition, the corpora lutea of all ovaries from pregnant females were counted at necropsy. The uterine implantation sites were counted. In the case of the non-pregnant female, the procedure was enhanced by staining the uteri with a 1% ammonium polysulphide solution.

ORGAN WEIGHTS: The following organs, removed from the five selected males and parental females from each group that were killed at the end of the study, were dissected free from fat and weighed before fixation. The reproductive organs were weighed from all animals that were killed at the end of the study: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus.

HISTOPATHOLOGY: Yes, samples of the following tissues from five males and five females from control and 1000 mg/kg/day dose group and those animals dying during the study: Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Caecum, Colon, Duodenum, Eyes, Gross lesions, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (cervical and mesenteric), Mammary gland, Muscle (skeletal), Pancreas, Oesophagus, Rectum, Salivary glands (submaxillary), Sciatic nerve, Skin (hind limb), Spinal cord (cervical, thoracic and lumbar), Spleen, Stomach, Thyroid/parathyroid, Trachea, Thymus, Urinary bladder, Vagina. The following tissues were examined from all animals of control and 1000 mg/kg/day dose group: Coagulating gland, Epididymides, Ovaries, Pituitary, Prostate, Seminal vesicles, Testes, Uterus/Cervix. Since there were indications of treatment-related changes in the stomach, examination was subsequently extended to include similarly prepared sections of the stomach from five selected males and females from the 250 and 50 mg/kg/day dose groups.

Postmortem examinations (offspring):

- The F1 offspring were sacrificed at 5 days of age.
- The animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain, litter weights, offspring bodyweights and quantitative functional performance data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney 'U' test. The non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers. The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value.

Reproductive indices:

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

Gestation and Parturition Data
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.
1) Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
2) Parturition Index: Parturition Index (%) = (Number of females delivering live offspring/Number of pregnant females) 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).
1) 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] x 100; % post-implantation loss = [(Number of implantation sites - number of offspring) / Number of implantation sites] x 100
2) 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) x 100;
3) Sex Ratio (% males): Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula: Sex Ratio (% males) = (Number of male offspring / Number of offspring of determined sex) x 100.

MORTALITY: There were no deaths related to treatment. One female treated with 250 mg/kg/day was killed in extremis on gestation Day 24, following difficulties encountered during parturition. In isolation and in the absence of any histopathological correlates, this death was considered unrelated to treatment.

CLINICAL SIGNS: No clinically observable signs of toxicity were detected. Treatment related effects were confined to sporadic episodes of increased salivation detected immediately after dosing for animals of either sex treated with 1000 mg/kg/day from Day 4. Incidents of generalised red/brown staining and wet fur (males only) were also detected for these animals, with red/brown staining also detected for females treated with 250 mg/kg/day. In addition incidents of noisy respiration were detected for seven males and three females treated with 1000 mg/kg/day and for one male and one female treated with 250 mg/kg/day. These observations are often reported following the oral administration of a slightly unpalatable or irritant test material formulation, and in isolation, are considered to be of no toxicological importance. The interim death female treated with 250 mg/kg/day displayed pilo-erection, tip-toe gait and hunched posture during parturition, the severity of these observations subsequently led to the female being killed in extremis. The observations detected were due to difficulties encountered during parturition. As this was an isolated incident and in the absence of a dose related response this death was considered unrelated to treatment.

BODY WEIGHT AND WEIGHT GAIN: No adverse effect on bodyweight change was detected for treated males in comparison to controls throughout the treatment period, or for females throughout the maturation, gestation or lactation phases of the study.

FOOD CONSUMPTION: A statistically significant reduction in dietary intake was evident for females treated with 1000 mg/kg/day during lactation when compared to controls. No adverse effect on dietary intake was detected for males throughout the treatment period, or for females during maturation and gestation.

WATER CONSUMPTION: Daily visual inspection of water bottles revealed no intergroup differences.

HAEMATOLOGY: No treatment-related haematological changes were detected prior to mating or at termination.

CLINICAL CHEMISTRY: No treatment-related changes were detected in the blood parameters measured. A statistically significant increase in albumin/globulin ratio was detected for males treated with 1000 mg/kg/day at Day 14 and termination. The apparent increase was, in reality, due to a single low control value, and in the absence of related changes in levels of total protein and albumin, was considered unrelated to test material toxicity. Males treated with 1000 mg/kg/day also showed a statistical significant decrease in levels of calcium ions and inorganic phosphate at Day 14. As decreases in these parameters were not detected at the terminal bleeds these changes were considered to be incidental and not related to test material toxicity. Males of this treatment group at termination also showed a significant increase in alkaline phosphatase. The increase in alkaline phosphatase was of minimal statistical significance, and was a consequence of a single low value in the controls therefore, was considered unrelated to test material toxicity. A statistically significant decrease in plasma urea was detected for males treated with 1000 or 250 mg/kg/day. The significance was p<0.05 and p
NEUROBEHAVIOUR: Weekly open field arena observations did not reveal any treatment-related effects during the study. All inter and intra group variations in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance. No treatment-related effects were detected in the functional performance parameters investigated. Statistical analysis of the data did not reveal any significant inter group differences. No treatment-related effects were detected in sensory reactivity. All inter and intra group differences in sensory reactivity scores were considered to be result of normal variation for rats of the strain and age used and were of no toxicological importance.

ORGAN WEIGHTS: No treatment related effects were detected. Liver weights relative to terminal bodyweight were elevated for animals of either sex treated with 1000 mg/kg/day. In the absence of relating histopathological evidence to suggest damage or impaired function of the liver, this finding was considered unrelated to test material toxicity.

GROSS PATHOLOGY: The incidental macroscopic observations detected included hydronepherosis of the right kidney for a single male treated with 1000 mg/kg/day. One male treated with 250 mg/kg/day had dark contents of the bladder; and one male treated with 50 mg/kg/day had a red mass. This mass was approximately 5mm by - 10mm in size and attached to the fat surrounding the epididymis. One control male had crystalline solids in the bladder, the bladder was also distended. All these findings were considered incidental, and in the absence of a dose related response, were considered unrelated to treatment. One control male had a damaged left eye, this was a physical injury that occurred during the necropsy procedure and so was unrelated to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC: Acanthosis, occasionally with associated hyperkeratosis, was observed in relation to treatment in the forestomach among animals of either sex treated with 1000 mg/kg/day. Such changes were also observed for one female treated with 250 mg/kg/day and also in the premature death animal from this treatment level. Although such changes in the forestomach do occasionally occur spontaneously in untreated laboratory maintained rats, a relationship to treatment at this dose level cannot be reliably excluded. There was one unscheduled death during the course of the study. There was peripheral inflammation of the uterus.

MATING: No adverse effect on mating performance, fertility or gestation lengths was detected. All animals mated within four days of pairing (at their first oestrus opportunity). One female treated with 1000 mg/kg/day mated within four days of pairing but failed to achieve pregnancy. This is a low incidence finding in reproductive studies and as this was a single incident, was considered to be of no toxicological significance.

Dose descriptor:

NOEL

Remarks:

General toxicity

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Histopathology of the stomach: Acanthosis, occasionally with associated hyperkeratosis was observed in relation to treatment in the forestomach among animals treated with 1000 mg/kg/day.

Dose descriptor:

NOEL

Remarks:

General toxicity

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Histopathology of the stomach: Acanthosis, occasionally with associated hyperkeratosis was observed in relation to treatment in the forestomach among females treated with 1000 mg/kg/day and 250 mg/kg/day.

Dose descriptor:

NOEL

Remarks:

Fertility

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed on fertility.

VIABILITY (OFFSPRING)
Following birth, offspring survival was reduced for litters from females treated with 1000 mg/kg/day with lower values for live birth index and viability index observed for high dose females when compared to controls.

CLINICAL SIGNS (OFFSPRING)
At 1000 mg/kg/day fourteen offspring from seven litters were either missing or found dead at Day 1. In addition nine other pup deaths were observed throughout the remaining days of lactation. One litter from the 1000 mg/kg/day treatment group showed distended dark abdomens throughout the lactation period and another two litters from this treatment group had distended abdomens throughout the lactation period; one of these litters were also all cold on Day 1. Four other pups from the 1000 mg/kg/day treatment group were noted as cold, one on Day 3, two on Day 4 and one on Day 5. A lower number of offspring successfully demonstrated surface righting reflex at 1000 mg/kg/day compared with offspring from the control group, on Day 1 post partum.

BODY WEIGHT (OFFSPRING)
Offspring bodyweight at Day 1 of age was significantly lower for offspring of females treated with 1000 mg/kg/day; subsequent bodyweight at Day 4 and bodyweight gain to Day 4 was also significantly lower when compared to controls. The lower offspring bodyweight detected at 1000 mg/kg/day led to the lower litter weight at Day 1 and Day 4 lactation for offspring of females of this treatment group when compared to controls.

GROSS PATHOLOGY (OFFSPRING)
The only treatment related effect detected was distended abdomens for one litter of the 1000 mg/kg/day treatment group, due to gaseous distension of the stomach and intestines. No treatment-related macroscopic abnormalities were detected for the interim death or terminal kill offspring from the remaining litters of the 1000 mg/kg/day treatment group or for offspring from the 250 or 50 mg/kg/day treatment group. The incidental findings, consisting of autolytic changes, missing tail, small in size, dent in head and a wound, are low incident findings in newly born rodent offspring, and are unrelated to treatment.

OTHER FINDINGS (OFFSPRING)
The number of corpora lutea was similar in all groups, and the number of implantations was slightly higher in females treated with 1000 mg/kg/day so was considered to be unaffected by treatment. Sex ratio was similar in all groups and did not indicate any selective effect of treatment on the survival of either sex.

Dose descriptor:

NOEL

Remarks:

Developmental

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reduction in live birth index and viability index, lower offspring and litter weight, whilst three litters at this dose level also had gaseous distension of the abdomen.

Reproductive effects observed:

not specified

DISCUSSION

Treatment related clinical observations at 250 and 1000 mg/kg/day were confined to increased salivation, noisy respiration and generalised red/brown staining. These observations are often associated with the oral administration of an unpalatable or irritant test material formulation and are supported by the irritation detected in the stomach during histopathological examination. Microscopic investigations revealed acanthosis of the stomach, occasionally with associated hyperkeratosis of the forestomach for animals of either sex treated with 1000 mg/kg/day; for one terminal kill female and the interim death female treated with 250 mg/kg/day. However, in the absence of necrosis or degenerative changes to the stomach, this finding was considered not to represent an effect of systemic toxicity. Bodyweight change and dietary intake was not adversely affected for males throughout the study or for females throughout maturation and gestation, however, a lower dietary intake was observed for females treated with the highest dose during lactation. This was considered to result from the irritation detected in the stomach. Treatment related reproductive findings included a reduction in the live birth index and in the viability index for offspring treated with 1000 mg/kg/day. A number of offspring from this treatment group were missing or found dead at Day 1 of lactation additionally nine other pups from this treatment group died during lactation, this is thought to be a consequence of the impaired physical health of the females caused by the irritation of the stomach. This led to the low bodyweight and litter weight of offspring of the 1000 mg/kg/day treatment group at Day 1 and Day 4. Additionally, a lower number of offspring successfully demonstrated surface righting reflex, compared with controls on Day 1 post partum, this is considered to be due to the physically weaker and smaller offspring at 1000 mg/kg/day when compared to controls. Three litters from this treatment group had distension of the abdomen throughout the lactation period with one of these litters having gaseous distension of the stomach and intestines at necropsy.

Conclusions:

Treatment-related effects detected in the stomach for animals of either sex treated with 1000 mg/kg/day and for females treated with 250 mg/kg/day. Treatment-related effects on reproduction were observed at 1000 mg/kg/day. These included a reduction in the live birth index and viability index; lower offspring and litter weight, whilst three litters at this dose level also had gaseous distension of the abdomen. Therefore the NOEL for general toxicity was considered to be 250 and 50 mg/kg/day for males and females, respectively. The NOEL for fertility and and developmental toxicity was considered to be 1000 and 250 mg/kg/day.

Executive summary:

In a GLP compliant combined repeat dose toxicity study with reproduction/developmental toxicity screening test, performed according to OECD Guideline 422, the test substance was administered by gavage to three groups each of ten male and ten female Sprague-Dawley rats, for up to forty-six consecutive days (during 2 weeks premating, mating, gestation and up to day 4 of lactation), at dose levels of 50, 250 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Polyethylene glycol 400). Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating on five selected non-recovery males and females from each dose group, and again at termination. Pairing of animals within each dose group was undertaken on a one male: one female basis on Day 15 of the study, to produce litters. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of reflexological response. Extensive functional observations were performed on five selected parental males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4 post partum. Males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination, organ weights and histopathological evaluation of selected tissues was performed.

No treatment-related effects were observed on mortality, clinical signs, behavioural assessments, functional performance tests, sensory reactivity assessments, bodyweights, food consumption of males, water consumption, haematology, blood chemistry, macroscopic observations at necropsy, and organ weights. Females treated with 1000 mg/kg/day showed a slight reduction in food consumption during lactation when compared to controls, but no adverse effect on dietary intake was detected for females during the maturation or gestation phases of the study. Histopathological examination revealed a treatment­related change in the stomach: Acanthosis, occasionally with associated hyperkeratosis, was observed in relation to treatment in the forestomach among animals of either sex treated with 1000 mg/kg/day. Such changes were also observed for one female treated with 250 mg/kg/day and also in the premature death female animal from this treatment level.

No adverse effects on mating performance, fertility or gestation were detected.

Females treated with 1000 mg/kg/day showed a lower live birth and viability index in comparison to controls, due to the birth of less offspring per litter and a lower survival rate for offspring during the lactation period. At 1000 mg/kg/day fourteen pups were either missing or found dead at Day 1 of lactation. In addition nine other deaths of pups were observed throughout the remaining days of lactation. Offspring from the 1000 mg/kg/day dose group showed less successful completion of surface righting assessments. In addition lower total litter weights and lower offspring bodyweight was detected at this dose level. These effects are thought to be a consequence of the impaired physical health of the females caused by the irritation of the stomach. Finally, three litters from this treatment group had distension of the abdomen throughout the lactation period with one of these litters having gaseous distension of the stomach and intestines at necropsy.

In conclusion, the oral administration of the test substance to rats by gavage, at dose levels of 50, 250 and 1000 mg/kg/day, resulted in treatment-related effects detected in the stomach for animals of either sex treated with 1000 mg/kg/day and for females treated with 250 mg/kg/day. The No Observed Effect Level (NOEL) for parental toxicity was considered to be 250 mg/kg/day for males and 50 mg/kg/day for females. Treatment-related effects on reproduction were observed at 1000 mg/kg/day. These were confined to a reduction in the live birth index and viability index. In addition lower offspring and litter weight were detected whilst three litters at this dose level also had gaseous distension of the abdomen. No such effects on reproduction were detected at 250 mg/kg/day, therefore, the NOEL for fertility and developmental toxicity was considered to be 1000 and 250 mg/kg/day, respectively.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP-compliant guideline study, klimisch 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a GLP compliant combined repeat dose toxicity study with reproduction/developmental toxicity screening test, performed according to OECD Guideline 422, the test substance was administered by gavage to three groups each of ten male and ten female Sprague-Dawley rats, for up to forty-six consecutive days (during 2 weeks premating, mating, gestation and up to day 4 of lactation), at dose levels of 50, 250 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Polyethylene glycol 400). Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating on five selected non-recovery males and females from each dose group, and again at termination. Pairing of animals within each dose group was undertaken on a one male: one female basis on Day 15 of the study, to produce litters. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of reflexological response. Extensive functional observations were performed on five selected parental males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4 post partum. Males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination, organ weights and histopathological evaluation of selected tissues was performed.

No treatment-related effects were observed on mortality, clinical signs, behavioural assessments, functional performance tests, sensory reactivity assessments, bodyweights, food consumption of males, water consumption, haematology, blood chemistry, macroscopic observations at necropsy, and organ weights. Females treated with 1000 mg/kg/day showed a slight reduction in food consumption during lactation when compared to controls, but no adverse effect on dietary intake was detected for females during the maturation or gestation phases of the study. Histopathological examination revealed a treatment­related change in the stomach: Acanthosis, occasionally with associated hyperkeratosis, was observed in relation to treatment in the forestomach among animals of either sex treated with 1000 mg/kg/day. Such changes were also observed for one female treated with 250 mg/kg/day and also in the premature death female animal from this treatment level.

No adverse effects on mating performance, fertility or gestation were detected.

Females treated with 1000 mg/kg/day showed a lower live birth and viability index in comparison to controls, due to the birth of less offspring per litter and a lower survival rate for offspring during the lactation period. At 1000 mg/kg/day fourteen pups were either missing or found dead at Day 1 of lactation. In addition nine other deaths of pups were observed throughout the remaining days of lactation. Offspring from the 1000 mg/kg/day dose group showed less successful completion of surface righting assessments. In addition lower total litter weights and lower offspring bodyweight was detected at this dose level. These effects are thought to be a consequence of the impaired physical health of the females caused by the irritation of the stomach. Finally, three litters from this treatment group had distension of the abdomen throughout the lactation period with one of these litters having gaseous distension of the stomach and intestines at necropsy.

In conclusion, the oral administration of the test substance to rats by gavage, at dose levels of 50, 250 and 1000 mg/kg/day, resulted in treatment-related effects detected in the stomach for animals of either sex treated with 1000 mg/kg/day and for females treated with 250 mg/kg/day. The No Observed Effect Level (NOEL) for parental toxicity was considered to be 250 mg/kg/day for males and 50 mg/kg/day for females. Treatment-related effects on reproduction were observed at 1000 mg/kg/day. These were confined to a reduction in the live birth index and viability index. In addition lower offspring and litter weight were detected whilst three litters at this dose level also had gaseous distension of the abdomen. No such effects on reproduction were detected at 250 mg/kg/day, therefore, the NOEL for fertility and developmental toxicity was considered to be 1000 and 250 mg/kg/day, respectively.

Short description of key information:
The NOEL for fertility (OECD 422, GLP) in rats is determined to be 1000 mg/kg bw, the highest dose tested.

Justification for selection of Effect on fertility via oral route:
Only study available, GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Effects on developmental toxicity

Description of key information

The NOEL in rats is determined (OECD 422, GLP) to be 50 and 250 mg/kg bw for maternal and developmental toxicity, respectively.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

SafePharm Laboratories Ltd., Shardlow Business Park, London Road, Shardlow, Derby, DE72 2GD, UK

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, Kent
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: males 250-305 g, females 169-210 g.
- Fasting period before study: No
- Housing: Initially, all animals were housed in groups of five in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper. During the mating phase, animals were transferred to similar cages on a one male:one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes. Environmental enrichment was provided in the form of wooden chew blocks (B&K Universal Ltd, Hull, UK) and cardboard fun tunnels (Datesand Ltd, Cheshire, UK) except for mated females during gestation and lactation.
- Diet: A pelleted diet (Rodent PMI 5002 (Certified) diet, BCM IPS Ltd., London, UK, ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ±2
- Humidity (%): 55±15
- Air changes (per hr): ≥15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was prepared at the appropriate concentrations as a solution in Polyethylene glycol 400. The stability and homogeneity of the test material formulations were determined. The formulations were stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4°C in the dark.

VEHICLE
- Amount of vehicle: 4 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test material formulation were taken on a weekly basis and analysed for concentration of 3,4-Dimethylbenzaldehyde. The concentration of 3, 4-Dimethyl Benzaldehyde in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique. The test material formulations were diluted with acetonitrile to give a final, theoretical test material concentration of approximately 0.05 mg/mL. Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 0.05 mg/mL. For homogeneity determinations, the test material formulations were mixed thoroughly and. samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate. For stability determinations, the test material formulations were sampled and analysed initially and then after storage at approximately +4°C in the dark for fourteen days. The results indicate that the analysed formulations were within acceptable limits for the purpose of this study.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually during the period of gestation and lactation

Duration of treatment / exposure:

up to forty-six consecutive days

Frequency of treatment:

daily

Duration of test:

From 2 weeks before mating, until day 5 post partum.

Remarks:

Doses / Concentrations:
50, 250, 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on preliminary range-finder in the rat.
- Chronological Sequence of Study: Groups of ten male and ten female animals were treated daily (from Day 1 of the study) at the appropriate dose level throughout the study (except for females during parturition where applicable). Prior to the start of treatment and once weekly, all animals were observed for signs of functional/behavioural toxicity. One day prior to pairing (Day 14), blood samples were taken from five males and five females, randomly selected from each dose group and analysed for haematological and blood chemical assessment. Blood samples were also taken at termination from five males and five females and analysed for haematological and blood chemical assessment. On Day 15, all animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days. Following evidence of mating using vaginal smearing, the males were returned to their original cages and females were transferred to individual cages. On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean pup weight, clinical observations and reflexological response were also performed during this period. At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli. Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically. At Day 5 post partum, all surviving females and offspring were killed and examined macroscopically.

Maternal examinations:

CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
- All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before and after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before and after dosing, and one hour after dosing at weekends (except for females during parturition where applicable).

BODY WEIGHT: Yes
- Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly until mating was evident. Bodyweights were then recorded on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, determined as g/animal/day
- During the maturation period, weekly food consumption was recorded for each cage of adults. For females showing evidence of mating, food consumption was recorded during the period of gestation. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Food efficiency (the ratio of bodyweight change/dietary intake) was calculated retrospectively during maturation and the first two weeks of gestation. Due to offspring growth and milk production, food efficiency could not be accurately calculated during the final week of gestation and during lactation.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 14 (day prior to pairing) and at termination
- Animals fasted: No
- How many animals: 5 females of each test and control group
- Parameters examined: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), Erythrocyte indices: [mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC)], Total leucocyte count (WBC), Differential leucocyte count: [neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas), Reticulocyte count (Retic; cresyl blue stained slides were prepared but reticulocytes were not assessed)], Platelet count (PLT), Prothrombin time (CT), Activated thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 14 (day prior to pairing) and at termination
- Animals fasted: No
- How many animals: 5 females of each test and control group
- Parameters examined: Urea, Glucose, Total protein, Albumin, Albumin/Globulin (A/G) ratio (by calculation), Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium(Ca++), Inorganic phosphorus (P), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Alkaline phosphatase (AP), Creatinine, Total cholesterol, Total bilirubin.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected females per dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
- Dose groups that were examined: all
- Battery of functions tested: behavioural assessment, functional performance tests (motor activity, Forelimb/Hindlimb Grips Strength), Sensory reactivity

GROSS PATHOLOGY: Yes, a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS: The following organs, removed from the five selected parental females from each group that were killed at the end of the study, were dissected free from fat and weighed before fixation. The reproductive organs were weighed from all animals that were killed at the end of the study: Adrenals, Brain, Heart, Kidneys, Liver, Ovaries, Spleen, Thymus.

HISTOPATHOLOGY: Yes, samples of the following tissues from five females from control and 1000 mg/kg/day dose group and those animals dying during the study: Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Caecum, Colon, Duodenum, Eyes, Gross lesions, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (cervical and mesenteric), Mammary gland, Muscle (skeletal), Pancreas, Oesophagus, Rectum, Salivary glands (submaxillary), Sciatic nerve, Skin (hind limb), Spinal cord (cervical, thoracic and lumbar), Spleen, Stomach, Thyroid/parathyroid, Trachea, Thymus, Urinary bladder, Vagina. The following tissues were examined from all animals of control and 1000 mg/kg/day dose group: Coagulating gland, Ovaries, Pituitary, Uterus/Cervix. Since there were indications of treatment-related changes in the stomach, examination was subsequently extended to include similarly prepared sections of the stomach from five females from the 250 and 50 mg/kg/day dose groups.

Ovaries and uterine content:

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

Fetal examinations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number of offspring born, number and sex of pups alive recorded daily and reported on Day 1 and 4 post partum, clinical condition, weight gain, reflexological response (surface righting reflex).
The F1 offspring were sacrificed at 5 days of age. The animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain, litter weights, offspring bodyweights and quantitative functional performance data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney 'U' test. The non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers. The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value.

Indices:

1) Pre-coital Interval, Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
2) Fertility Indices (calculated per group): Mating Index (%) = (Number of animals mated/Number of animals paired) x 100; Pregnancy Index (%) = (Number of pregnant females/Number of animals mated) x 100
3) Gestation Length: The number of days of gestation including the day for observation of mating and the start of parturition.
4) Parturition Index (%) = (Number of females delivering live offspring/Number of pregnant females) x 100
5) Implantation Losses (%): % pre-implantation loss = [(Number of Corpora Lutea - Number of implantation sites) / Number of corpora lutea] x 100; % post-implantation loss = [(Number of implantation sites - number of offspring) / Number of implantation sites] x 100
6) Live Birth and Viability Indices: 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) x 100
7) Sex Ratio (% males): Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula:; Sex Ratio (% males) = (Number of male offspring / Number of offspring of determined sex) x 100

Details on maternal toxic effects:

Details on maternal toxic effects:
MORTALITY: There were no deaths related to treatment. One female treated with 250 mg/kg/day was killed in extremis on gestation Day 24, following difficulties encountered during parturition. In isolation and in the absence of any histopathological correlates, this death was considered unrelated to treatment.

CLINICAL SIGNS: No clinically observable signs of toxicity were detected. Treatment related effects were confined to sporadic episodes of increased salivation detected immediately after dosing for animals of either sex treated with 1000 mg/kg/day from Day 4. Incidents of generalised red/brown staining and wet fur (males only) were also detected for these animals, with red/brown staining also detected for females treated with 250 mg/kg/day. In addition incidents of noisy respiration were detected for three females treated with 1000 mg/kg/day and for one female treated with 250 mg/kg/day. These observations are often reported following the oral administration of a slightly unpalatable or irritant test material formulation, and in isolation, are considered to be of no toxicological importance. The interim death female treated with 250 mg/kg/day displayed pilo-erection, tip-toe gait and hunched posture during parturition, the severity of these observations subsequently led to the female being killed in extremis. The observations detected were due to difficulties encountered during parturition. As this was an isolated incident and in the absence of a dose related response this death was considered unrelated to treatment.

BODY WEIGHT AND WEIGHT GAIN: No adverse effect on bodyweight change was detected for females throughout the maturation, gestation or lactation phases of the study.

FOOD CONSUMPTION: A statistically significant reduction in dietary intake was evident for females treated with 1000 mg/kg/day during lactation when compared to controls. No adverse effect on dietary intake was detected for females during maturation and gestation.

WATER CONSUMPTION: Daily visual inspection of water bottles revealed no intergroup differences.

HAEMATOLOGY: No treatment-related haematological changes were detected prior to mating or at termination.

CLINICAL CHEMISTRY: No treatment-related changes were detected in the blood parameters measured.

NEUROBEHAVIOUR: Weekly open field arena observations did not reveal any treatment-related effects during the study. All inter and intra group variations in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance. No treatment-related effects were detected in the functional performance parameters investigated. Statistical analysis of the data did not reveal any significant inter group differences. No treatment-related effects were detected in sensory reactivity. All inter and intra group differences in sensory reactivity scores were considered to be result of normal variation for rats of the strain and age used and were of no toxicological importance.

ORGAN WEIGHTS: No treatment related effects were detected. Liver weights relative to terminal bodyweight were elevated for animals of treated with 1000 mg/kg/day. In the absence of relating histopathological evidence to suggest damage or impaired function of the liver, this finding was considered unrelated to test material toxicity.

GROSS PATHOLOGY: No treatment-related changes were detected.

HISTOPATHOLOGY: NON-NEOPLASTIC: Acanthosis, occasionally with associated hyperkeratosis, was observed in relation to treatment in the forestomach among animals of treated with 1000 mg/kg/day. Such changes were also observed for one female treated with 250 mg/kg/day and also in the premature death animal from this treatment level. Although such changes in the forestomach do occasionally occur spontaneously in untreated laboratory maintained rats, a relationship to treatment at this dose level cannot be reliably excluded. There was one unscheduled death during the course of the study. There was peripheral inflammation of the uterus.

MATING: No adverse effect on mating performance, fertility or gestation lengths was detected. All animals mated within four days of pairing (at their first oestrus opportunity). One female treated with 1000 mg/kg/day mated within four days of pairing but failed to achieve pregnancy. This is a low incidence finding in reproductive studies and as this was a single incident, was considered to be of no toxicological significance.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING)
Following birth, offspring survival was reduced for litters from females treated with 1000 mg/kg/day with lower values for live birth index and viability index observed for high dose females when compared to controls.

CLINICAL SIGNS (OFFSPRING)
At 1000 mg/kg/day fourteen offspring from seven litters were either missing or found dead at Day 1. In addition nine other pup deaths were observed throughout the remaining days of lactation. One litter from the 1000 mg/kg/day treatment group showed distended dark abdomens throughout the lactation period and another two litters from this treatment group had distended abdomens throughout the lactation period; one of these litters were also all cold on Day 1. Four other pups from the 1000 mg/kg/day treatment group were noted as cold, one on Day 3, two on Day 4 and one on Day 5. A lower number of offspring successfully demonstrated surface righting reflex at 1000 mg/kg/day compared with offspring from the control group, on Day 1 post partum.

BODY WEIGHT (OFFSPRING)
Offspring bodyweight at Day 1 of age was significantly lower for offspring of females treated with 1000 mg/kg/day; subsequent bodyweight at Day 4 and bodyweight gain to Day 4 was also significantly lower when compared to controls. The lower offspring bodyweight detected at 1000 mg/kg/day led to the lower litter weight at Day 1 and Day 4 lactation for offspring of females of this treatment group when compared to controls.

GROSS PATHOLOGY (OFFSPRING)
The only treatment related effect detected was distended abdomens for one litter of the 1000 mg/kg/day treatment group, due to gaseous distension of the stomach and intestines. No treatment-related macroscopic abnormalities were detected for the interim death or terminal kill offspring from the remaining litters of the 1000 mg/kg/day treatment group or for offspring from the 250 or 50 mg/kg/day treatment group. The incidental findings, consisting of autolytic changes, missing tail, small in size, dent in head and a wound, are low incident findings in newly born rodent offspring, and are unrelated to treatment.

OTHER FINDINGS (OFFSPRING)
The number of corpora lutea was similar in all groups, and the number of implantations was slightly higher in females treated with 1000 mg/kg/day so was considered to be unaffected by treatment. Sex ratio was similar in all groups and did not indicate any selective effect of treatment on the survival of either sex.

Abnormalities:

not specified

Developmental effects observed:

not specified

DISCUSSION

Treatment related clinical observations at 250 and 1000 mg/kg/day were confined to increased salivation, noisy respiration and generalised red/brown staining. These observations are often associated with the oral administration of an unpalatable or irritant test material formulation and are supported by the irritation detected in the stomach during histopathological examination. Microscopic investigations revealed acanthosis of the stomach, occasionally with associated hyperkeratosis of the forestomach for animals of either sex treated with 1000 mg/kg/day; for one terminal kill female and the interim death female treated with 250 mg/kg/day. However, in the absence of necrosis or degenerative changes to the stomach, this finding was considered not to represent an effect of systemic toxicity. Bodyweight change and dietary intake was not adversely affected for males throughout the study or for females throughout maturation and gestation, however, a lower dietary intake was observed for females treated with the highest dose during lactation. This was considered to result from the irritation detected in the stomach. Treatment related reproductive findings included a reduction in the live birth index and in the viability index for offspring treated with 1000 mg/kg/day. A number of offspring from this treatment group were missing or found dead at Day 1 of lactation additionally nine other pups from this treatment group died during lactation, this is thought to be a consequence of the impaired physical health of the females caused by the irritation of the stomach. This led to the low bodyweight and litter weight of offspring of the 1000 mg/kg/day treatment group at Day 1 and Day 4. Additionally, a lower number of offspring successfully demonstrated surface righting reflex, compared with controls on Day 1 post partum, this is considered to be due to the physically weaker and smaller offspring at 1000 mg/kg/day when compared to controls. Three litters from this treatment group had distension of the abdomen throughout the lactation period with one of these litters having gaseous distension of the stomach and intestines at necropsy.

Conclusions:

Treatment-related effects were detected in the stomach for females treated with 250 and 1000 mg/kg/day. Treatment-related effects on reproduction were observed at 1000 mg/kg/day. These included a reduction in the live birth index and viability index; lower offspring and litter weight, whilst three litters at this dose level also had gaseous distension of the abdomen. Therefore, the NOEL for maternal and developmental toxicity was considered to be 50 and 250 mg/kg/day, respectively

Executive summary:

In a GLP compliant combined repeat dose toxicity study with reproduction/developmental toxicity screening test, performed according to OECD Guideline 422, the test substance was administered by gavage to three groups each of ten male and ten female Sprague-Dawley rats, for up to forty-six consecutive days (during 2 weeks premating, mating, gestation and up to day 4 of lactation), at dose levels of 50, 250 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Polyethylene glycol 400). Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating on five selected non-recovery males and females from each dose group, and again at termination. Pairing of animals within each dose group was undertaken on a one male: one female basis on Day 15 of the study, to produce litters. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of reflexological response. Extensive functional observations were performed on five selected parental females from each dose group on Day 4 post partum. Females and offspring were terminated on Day 5 post partum. All animals were subjected to a gross necropsy examination, organ weights and histopathological evaluation of selected tissues was performed.

No treatment-related effects were observed on mortality, clinical signs, behavioural assessments, functional performance tests, sensory reactivity assessments, bodyweights, food consumption of males, water consumption, haematology, blood chemistry, macroscopic observations at necropsy, and organ weights. Females treated with 1000 mg/kg/day showed a slight reduction in food consumption during lactation when compared to controls, but no adverse effect on dietary intake was detected for females during the maturation or gestation phases of the study. Histopathological examination revealed a treatment­related change in the stomach: Acanthosis, occasionally with associated hyperkeratosis, was observed in relation to treatment in the forestomach among animals treated with 1000 mg/kg/day. Such changes were also observed for one female treated with 250 mg/kg/day and also in the premature death female animal from this treatment level.

No adverse effects on mating performance, fertility or gestation were detected.

Females treated with 1000 mg/kg/day showed a lower live birth and viability index in comparison to controls, due to the birth of less offspring per litter and a lower survival rate for offspring during the lactation period. At 1000 mg/kg/day fourteen pups were either missing or found dead at Day 1 of lactation. In addition nine other deaths of pups were observed throughout the remaining days of lactation. Offspring from the 1000 mg/kg/day dose group showed less successful completion of surface righting assessments. In addition lower total litter weights and lower offspring bodyweight was detected at this dose level. These effects are thought to be a consequence of the impaired physical health of the females caused by the irritation of the stomach. Finally, three litters from this treatment group had distension of the abdomen throughout the lactation period with one of these litters having gaseous distension of the stomach and intestines at necropsy.

In conclusion, the oral administration of the test substance to rats by gavage, at dose levels of 50, 250 and 1000 mg/kg/day, resulted in treatment-related effects detected in the stomach for females treated with 250 and 1000 mg/kg/day. The No Observed Effect Level (NOEL) for maternal toxicity was considered to be 50 mg/kg/day. Treatment-related effects on reproduction were observed at 1000 mg/kg/day. These were confined to a reduction in the live birth index and viability index. In addition lower offspring and litter weight were detected whilst three litters at this dose level also had gaseous distension of the abdomen. No such effects on reproduction were detected at 250 mg/kg/day, therefore, the NOEL for developmental toxicity was considered to be 250 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant OECD guideline study, klimisch 1

Additional information

In a GLP compliant combined repeat dose toxicity study with reproduction/developmental toxicity screening test, performed according to OECD Guideline 422, the test substance was administered by gavage to three groups each of ten male and ten female Sprague-Dawley rats, for up to forty-six consecutive days (during 2 weeks premating, mating, gestation and up to day 4 of lactation), at dose levels of 50, 250 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Polyethylene glycol 400). Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating on five selected non-recovery males and females from each dose group, and again at termination. Pairing of animals within each dose group was undertaken on a one male: one female basis on Day 15 of the study, to produce litters. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of reflexological response. Extensive functional observations were performed on five selected parental females from each dose group on Day 4 post partum. Females and offspring were terminated on Day 5 post partum. All animals were subjected to a gross necropsy examination, organ weights and histopathological evaluation of selected tissues was performed.

No treatment-related effects were observed on mortality, clinical signs, behavioural assessments, functional performance tests, sensory reactivity assessments, bodyweights, food consumption of males, water consumption, haematology, blood chemistry, macroscopic observations at necropsy, and organ weights. Females treated with 1000 mg/kg/day showed a slight reduction in food consumption during lactation when compared to controls, but no adverse effect on dietary intake was detected for females during the maturation or gestation phases of the study. Histopathological examination revealed a treatment­related change in the stomach: Acanthosis, occasionally with associated hyperkeratosis, was observed in relation to treatment in the forestomach among animals treated with 1000 mg/kg/day. Such changes were also observed for one female treated with 250 mg/kg/day and also in the premature death female animal from this treatment level.

No adverse effects on mating performance, fertility or gestation were detected.

Females treated with 1000 mg/kg/day showed a lower live birth and viability index in comparison to controls, due to the birth of less offspring per litter and a lower survival rate for offspring during the lactation period. At 1000 mg/kg/day fourteen pups were either missing or found dead at Day 1 of lactation. In addition nine other deaths of pups were observed throughout the remaining days of lactation. Offspring from the 1000 mg/kg/day dose group showed less successful completion of surface righting assessments. In addition lower total litter weights and lower offspring bodyweight was detected at this dose level. These effects are thought to be a consequence of the impaired physical health of the females caused by the irritation of the stomach. Finally, three litters from this treatment group had distension of the abdomen throughout the lactation period with one of these litters having gaseous distension of the stomach and intestines at necropsy.

In conclusion, the oral administration of the test substance to rats by gavage, at dose levels of 50, 250 and 1000 mg/kg/day, resulted in treatment-related effects detected in the stomach for females treated with 250 and 1000 mg/kg/day. The No Observed Effect Level (NOEL) for maternal toxicity was considered to be 50 mg/kg/day. Treatment-related effects on reproduction were observed at 1000 mg/kg/day. These were confined to a reduction in the live birth index and viability index. In addition lower offspring and litter weight were detected whilst three litters at this dose level also had gaseous distension of the abdomen. No such effects on reproduction were detected at 250 mg/kg/day, therefore, the NOEL for developmental toxicity was considered to be 250 mg/kg/day.

Justification for selection of Effect on developmental toxicity: via oral route:
Only study available, GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Justification for classification or non-classification

Based on the absence of adverse effects on fertility and of developmental toxicity in the absence of parental toxicity, classification of the test material for effects on fertility or developmental toxicity is not warranted in accordance with EU Directive 67/548/EEC and EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.