Hatcol 1760

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 May 2008 to 16 December 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 21 August 2007 Date of Signature: 15 October 2007

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): Hatcol 1760
- Substance type: Clear colourless viscous liquid
- Physical state: liquid
- Lot/batch No.: A25983
- Storage condition of test material: room temperature in the dark.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, UK.
- Age at study initiation: 12 weeks of age.
- Weight at study initiation: The males weighed 281 to 350 g; the females weighed 187 to 235 g.
- Fasting period before study: No.
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Harlan Laboratories UK Ltd). During the mating phase, the non-recovery dose group animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. Recovery animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding.
- Diet (e.g. ad libitum): A pelleted diet Rodent 2018C Teklad Global Certified Diet (Harlan Laboratories U.K. Ltd, Oxon, UK) ad libitum. The diet was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Water (e.g. ad libitum): Mains drinking water ad libitum. The water was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Acclimation period: 12 days.

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

IN-LIFE DATES: From: Day 1 To: Day 43 (males); Day 1 To: Day 5 post partum (females).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The formulations of the test material was prepared at 50, 250 and 1000 mg/kg/day as a suspension in Arachis oil BP. The stability and homogeneity of the test material formulations were determined at the test laboratory. The formulations to be stable for at least 14 days.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Approximately 4ºC in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Analysis of a blank Arachis Oil BP produced no signal that interfered with the signal due to the test material.
- Amount of vehicle (if gavage): 4 ml/kg.
- Lot/batch no. (if required): Not reported.
- Purity: Not reported.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Summary; The concentration of Hatcol 1760 in the test material formulations was determined by gas chromatography (GC) using an external standard technique.
Materials and methods; The test material formulations were extracted with acetonitrile to give a final, theoretical test material concentration of approximately 0.1 mg/ml.
Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 0.1 mg/ml. The standard and sample solutions were analysed by GC.
The test material formulations were deemed homogeneous by visual inspection.
The test material formulations were sampled and analysed initially and then after storage at approximately +4ºC in the dark for 14 days.
The test material formulations were sampled and analysed within 3 days of preparation.

Results; A range of standard solutions covering the concentration range 0 to 0.2158 mg/ml, were prepared and analysed. The detector response was shown to be linear up to 0.2158 mg/ml.
Analysis of the solvent and a blank Arachis Oil BP (control) produced no signal that interfered with the signal due to the test material.
The analytical method has been considered to be sufficiently accurate for the purpose of this study. The test sample results have not been corrected for recovery.
Conclusion; The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.

Details on mating procedure:

i) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
ii) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.

Duration of treatment / exposure:

From: Day 1 To: Day 42 (males); Day 1 To: Day 4 of lpost partum (females).

Frequency of treatment:

Once daily.

Duration of test:

(IN-LIFE DATES): From: Day 1 To: Day 43 (males); Day 1 To: Day 5 post partum (females).

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
50 mg/kg/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
250 mg/kg/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10 animals per sex per dose and the control for non-recovery group.
5 animals per sex per dose (1000 mg/kg/day) and the control for recovery group.

Control animals:

yes, concurrent vehicle

Details on study design:

(Non-recovery group)
i) Groups of 10 male and 10 female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
iii) 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.
iv) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
v) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
vi) On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli and urinalysis was performed.
vii) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.
viii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
ix) Additional blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically.
x) Additional blood samples were taken from five randomly selected females from each dose group at termination for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all non-recovery females and surviving offspring were killed and examined macroscopically.

(Recovery group)
i) Groups of 5 male and 5 female rats were dosed according to dose group continuously up to the point of sacrifice of non-recovery males at which time treatment was discontinued.
ii) The males and females were maintained without treatment for a further 14 days.
iii) Urinalysis was performed for all males during the final week of recovery.
iv) Blood samples were taken for haematological and blood chemical assessment on Day 56.
v) After fourteen days of recovery, males and females were killed and examined macroscopically.

- Dose selection rationale: The dose levels were chosen based on the results of a preliminary range-finder performed as part of the study.
- Rationale for animal assignment (if not random): The rat was selected for this study as it is a readily available rodent species historically
used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
- Rationale for selecting satellite groups: Not reported.
- Post-exposure recovery period in satellite groups: 14 days.
- Section schedule rationale (if not random): Not applicable

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, up to 30 minutes after dosing, and 1 and 5 hours after
dosing, during the working week. Animals were observed immediately before dosing,
soon after dosing, and 1 hour after dosing at weekends (except for
females during parturition where applicable).
- Cage side observations checked: Overt signs of toxicity, ill-health and behavioural change.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, up to 30 minutes after dosing, and 1 and 5 hours after
dosing, during the working week. Animals were observed immediately before dosing,
soon after dosing, and 1 hour after dosing at weekends (except for
females during parturition where applicable).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for
males until termination and weekly for females until pairing. During the mating phase, females were weighed daily until mating was cofirmed. Bodyweights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Recovery animals were weighed on Day 1 (prior to dosing) and then weekly until termination.

FOOD CONSUMPTION: Yes
- During the maturation period, weekly food consumption was recorded for each cage of non-recovery adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-20 post coitum. For females with live litters, food consumption was recorded on Days 1 and 4 post partum. Weekly food consumptions were performed weekly for each cage of recovery adults throughout the study period.

FOOD EFFICIENCY: 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: Yes
- Time schedule for examinations: Daily. Water intake was observed by visual inspection of water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Haematological and blood chemical investigations were performed on five males and five females selected from each non-recovery test and control group on Day 14 (day prior to pairing) and at the end of the treatment period (Day 42/Day 4 post partum). In addition haematological and blood chemical investigations were performed on all recovery group animals after the 14 day treatment free period at termination (Day 56). Blood samples were obtained from the lateral tail vein at Day 14 and by cardiac puncture at termination.
- Anaesthetic used for blood collection: No.
- Animals fasted: No.
- How many animals: 5 males and 5 females selected from each test and control group in the non-recovery group. All animals (5 males and 5 females) in the recovery group.
- Parameters checked in tables 16 -19 which are attached were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: During the final week of treatment for non-recovery males and during the final week of the recovery period for recovery males.
- Metabolism cages used for collection of urine: Urine samples were collected overnight by housing the rats in metabolism cages.
- Animals fasted: Yes
- Parameters examined: The data in Table 20 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
(Behavioural assessment)
Prior to the start of treatment and at weekly intervals thereafter.
(Functional Performance Tests)
Prior to termination.
(Sensory Reactivity)
Prior to termination.

- Dose groups that were examined:
(Behavioural assessment)
All non-recovery animals.
(Functional Performance Tests)
Five selected males and females per non-recovery dose level.
(Sensory Reactivity)
Five selected males and females per non-recovery dose level.

- Parameters examined:
(Behavioural assessment)
Detailed individual clinical observations were performed for each animal using a purpose-built arena.
The following parameters were observed:
Gait, hyper/hypothermia, tremors, skin colour, twitches, respiration, convulsions, palpebral closure, bizarre/abnormal/stereotypic behaviour, urination, salivation, defecation, pilo-erection, transfer arousal, exophthalmia, tail elevation and lachrymation.
(Functional Performance Tests)
Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period).
Forelimb/Hindlimb Grip Strength. An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.
(Sensory Reactivity)
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed:
Grasp response, touch escape, vocalisation, pupil reflex, toe pinch, startle reflex, tail pinch, blink reflex and finger approach.

OTHER:
MATING
Non-recovery animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

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 and public holidays. The following was recorded for each female:
i) Date of mating
ii) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition
iv) Duration of gestation

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
- Other: parameters in Table 1 were examined.

Fetal examinations:

- External examinations: Yes (All offspring, including those dying offspring during the study)
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in offspring: Number of offspring born, number and sex of offspring alive recorded daily and reported on Day 1 and 4 post partum, clinical condition of offspring from birth to Day 5 post partum, individual offspring and litter weights on Day 1 and 4 post partum, physical Development and pathology.

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

GROSS EXAMINATION OF PUPS: All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

The following parameters were subjected to statistical analysis:
Quantitative functional performance data
Bodyweight and bodyweight change
Food consumption during gestation and lactation
Haematology, blood chemistry, absolute and bodyweight relative organ weights
Litter data
Sex ratio
Implantation losses and viability indices
Offspring bodyweight and bodyweight change
Offspring surface righting
The following statistical procedures were used:
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.
Non-parametric methods were used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
Probability values (p) are presented as follows:
p<0.001 ***
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.

i) Pre-coital Interval

Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii) 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.

i) Gestation Length

Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii) Parturition Index

The following was calculated for each group:

Parturition Index (%) = (Number of females delivering live offspring÷Number of pregnant females) ) x 100
Litter Responses

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).

i) 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 site)÷Number of corpora lutea] x 100

% post – implantation loss =[(Number of implantation sites - Total number of offspring born)÷ Number of implantation sites)] x 100

Historical control data:

Not reported.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
(Systematic toxicity)
Clinical signs and mortality; There were no treatment-related unscheduled deaths during the study. No toxicologically significant clinical signs were evident in terminal kill animals throughout the study. During the course of the study there were three mortalities (two males treated with 250 mg/kg/day and one control male). The two males treated with 250 mg/kg/day were killed in extremis after routine blood sampling on Day 42 and the control male was found dead on Day 43. An in-house investigation by our animal welfare experts revealed there to be a malfunction with the temperature control for the equipment used to warm the animals prior to blood sampling. The elevated temperatures in the equipment did not register as malfunctioning. The equipment was subsequently removed from use prior to it being repaired.

Body weight and and weight gain; No adverse effect on bodyweight gain was detected for males throughout the treatment period or for females throughout the two week maturation period. There were no adverse effects on bodyweight gains for females during the gestation or lactation phase of the study.

Food consumption; No adverse effect on food consumption was detected for males throughout the treatment period or for females throughout the two week maturation period. There were no adverse effects on food consumption for females during the gestation or lactation phase of the study.

Water consumption; Daily visual inspection of water bottles revealed no intergroup differences.

Haematology; Group mean values and standard deviations for test and control group animals are given in Table 16 below and Table 17 attached. There were no toxicologically significant changes in the haematological parameters measured.
Non-recovery males treated with 1000 mg/kg/day showed an increase in total leucocyte count and in lymphocyte count during the Day 14 assessments. Recovery 1000 mg/kg/day males also showed a statistically significant reduction in total leucocyte count during Day 56 assessments. In the absence of a similar effect seen at termination of treatment these intergroup differences were considered to be of no toxicological importance.

Clinical chemistry; Group mean values and standard deviations for test and control group animals are given in Table 18 and Table 19 attached. Non-recovery females treated with 1000 mg/kg/day showed an increase in cholesterol on Day 4 post partum.
No such effects were evident for non-recovery males treated with 1000 mg/kg/day, animals of either sex treated with 250 or 50 mg/kg/day or recovery animals following fourteen days without treatment. In the absence of a dose-related response the intergroup differences were considered of no toxicological significance.

Urinalysis; There were no treatment related changes in the urinalytical parameters measured.

Neurobehaviour; There were no treatment-related changes in the behavioural parameters and sensory reactivity measured. There were no toxicologically significant changes detected in the Functional Performance tested.

Gross pathology; No toxicologically significant macroscopic abnormalities were detected in terminal kill animals.

Organ weights; Group mean absolute and bodyweight-relative organ weight data are given in Tables 30 to 33 attached. Non-recovery animals of either sex treated with 1000 mg/kg/day showed a statistically significant increase in relative liver weight with females also showing a statistically significant increase in absolute liver weight. Relative kidney weight was increased for males from this treatment group. No such effects were detected in animals of either sex treated with 250 or 50 mg/kg/day or recovery animals following fourteen days without treatment.

Histopathology; Summary incidences of histopathological findings are given in Tables 34 and 35 attached. The following treatment-related changes were detected:
ADRENAL GLAND: Hypertrophy of the zona glomerulosa was seen in relation to treatment for males only at all treatment levels. This change is regarded as adaptive in nature and may be associated with renal changes.
The condition was observed to have regressed among Recovery 1000 mg/kg/day animals following an additional 14 days without treatment.
LIVER: Centrilobular hepatocyte enlargement was seen as a consequence of treatment for males treated with 1000 mg/kg/day but not at any other treatment level. There was no evidence of a similar effect for females.
The condition was observed to have regressed following completion of the recovery period.
Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive in nature.
KIDNEYS: Renal changes characterised by globular accumulations of eosinophilic material, tubular basophilia/degeneration, and tubular necrosis, were seen among males only treated with 1000 mg/kg/day. Globular accumulations of eosinophilic material were also seen for males treated with 250 mg/kg/day or at 50 mg/kg/day with tubular necrosis in a few males treated with 250 mg/kg/day. Similar accumulations were also demonstrated by examination of Mallory’s Heidenhain stained sections of kidney which assists in the diagnosis of but which is not diagnostic for α2-Microglobulin.
There was regression of globular accumulations of eosinophilic material among recovery 1000 mg/kg/day males following completion of the 14-day recovery period although residual tubular necrosis remained in four animals, possibly with an associated higher incidence/severity of isolated groups of basophilic tubules.
The presence of globular accumulations of eosinophilic material is consistent with hydrocarbon nephropathy, which results from the excessive accumulation of α2-microglobulin in renal proximal tubular epithelial cells. α2-Microglobulin is found only in the proximal tubular epithelium of adult males.
THYROID: Follicular cell hypertrophy was seen in relation to treatment for males treated with 1000 mg/kg/day, but not convincingly at any other treatment level. Females were not similarly affected. There was no difference in incidence or severity of the condition between recovery 1000 mg/kg/day and recovery control animals following completion of the recovery period.
THYMUS: Lymphoid atrophy was observed as a consequence of treatment for females treated with 1000 mg/kg/day but not at any other treatment level. Males were not similarly affected.
The condition was observed to have regressed among recovery group animals following completion of an additional fourteen days without treatment.

(Reproductive toxicity)
Mating; There were no treatment-related effects on mating performance.

Fertility; There were no treatment-related effects on fertility.

Gestation length; There were no significant intergroup differences in gestation lengths or parturition for treated females, with the majority of females giving birth following 22 to 24 days of gestation. The distribution for treated females was comparable to controls.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Remarks:

female

Effect level:

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

Basis for effect level:

other: other:

2

Dose descriptor:

NOAEL

Remarks:

male

Effect level:

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

Basis for effect level:

other: other:

3

Dose descriptor:

NOEL

Remarks:

male/female

Effect level:

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

Basis for effect level:

other: other:

4

Dose descriptor:

NOAEL

Remarks:

male/female

Effect level:

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

Basis for effect level:

other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Litter response; In total there were 9, 10, 8 and 10 females at control, 50, 250 and 1000 mg/kg/day respectively who gave birth to a live litter and successfully reared young to Day 5 of age. One female treated with 250 mg/kg/day gave birth to a live litter but had a total litter loss on Day 4. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

Litter size and viability; The mean numbers of corpora lutea observed for treated females did not indicate any adverse effect of treatment at 50, 250 or 1000 mg/kg/day. Subsequent litter size at Day 1 for treated animals was similar to controls. Post-natal survival was unaffected in all treated groups with litter size at Day 4 again being similar to controls.

Offspring growth and development; Group values for offspring bodyweight and bodyweight change are given in Tables 22 attached. Offspring bodyweight gain was slightly reduced from litters treated with 1000 mg/kg/day between Days 1 and 4 post partum.
The percentage of offspring who successfully showed surface righting reflex on Day 1 and the type, incidence and distribution of clinical signs in the offspring to termination on Day 5 of age was unaffected by maternal exposure.

Necropsy; No adverse effect of maternal treatment was observed.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The oral administration of Hatcol 1760 to rats by gavage, at dose levels of 50, 250 and 1000 mg/kg/day, resulted in treatment-related effect in males from all treatment groups and in females treated with 1000 mg/kg/day. A No Observed Effect Level (NOEL) has, therefore not been established for males however, no such changes were demonstrated at 250 mg/kg/day for females and the ‘No Observed Effect Level’ (NOEL) for females was, therefore considered to be 250 mg/kg/day.
The kidney changes identified histopathologically at 250 and 50 mg/kg/day were consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons. There is no evidence to suggest that the tubular necrosis seen in a few 250 mg/kg/day males is of independent origin since it is only seen in association with hydrocarbon nephropathy and was not observed among female rats. It is therefore considered to be associated with, or a consequence of, excessive accumulation of alpha-2-microglobulin in male rats in this investigation. This effect is, therefore, not indicative of a hazard to human health and, for the purposes of hazard evaluation, the “No Observed Adverse Effect Level” (NOAEL) for males, should be regarded as 250 mg/kg/day.
Offspring from litters treated with 1000 mg/kg/day showed a reduction in bodyweight gain between days 1 and 4 post partum. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was therefore considered to be 250 mg/kg/day.
The effects seen at 1000 mg/kg/day are minimal and because of higher litter size and the fact that there is offspring bodyweight gain albeit at a lower rate than controls, the NOAEL can be suggested as 1000 mg/kg/day.

Executive summary:

Summary of the study report;

Introduction.The study was designed to investigate the systemic toxicity and potential adverse effects of the test material on reproduction (including offspring development) and complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).

Methods. The test material was administered by gavage to three groups each of ten male and ten femaleWistar Han™:HsdRccHan™:WIST strain rats, for up to fifty-four consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), 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 (Arachis Oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for forty-two consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating and at termination on five selected non-recovery males and females from each dose group. In addition, haematology and blood chemistry were evaluated after the fourteen day treatment free period on all recovery animals. Urinalysis was evaluated on five selected non-recovery males at the end of the treatment period and for all recovery group males at the end of the treatment free period.

Pairing of animals within each non-recovery dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5post partum.

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.

Extensive functional observations were performed on five selected males from each non-recovery dose group after the completion of the mating phase, and for five selected parental females from each non-recovery dose group on Day 4post partum.

Surviving non-recovery males were terminated on Day 43, followed by the termination of all non-recovery females and offspring on Day 5post partum. All non-recovery animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Recovery animals were treated according to the dose group continuously up to the point of sacrifice of the non-recovery males at which time the treatment was discontinued. After fourteen days without treatment, the recovery males and females were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Adult Responses:

Mortality.There were no treatment-related unscheduled deaths during the study.

Clinical Signs.No toxicologically significant clinical signs were evident in terminal kill animals throughout the study.

Behavioural Assessment.There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests.There were no toxicologically significant changes in the functional performance parameters measured.

Sensory Reactivity Assessments.There were no treatment-related changes in sensory reactivity.

Bodyweights.No adverse effect on bodyweight development was detected.

Food Consumption and Food Efficiency.No adverse effect on dietary intake or food efficiency was detected.

Water Consumptions.No intergroup differences were detected.

Haematology.No toxicologically significant effects were detected in the haematological parameters measured.

Blood Chemistry.Non-recovery females treated with 1000 mg/kg/day showed an increase in cholesterol. No such effects were evident for non-recovery males treated with 1000 mg/kg/day, animals of either sex treated with 250 or 50 mg/kg/day or recovery animals following fourteen days without treatment.

Urinalysis.No treatment-related effect was detected in the urinalytical parameters measured.

Reproductive Performance:

Mating.There were no treatment-related effects on male or female mating or conception rates. The distribution of pre-coital intervals for treated animals was comparable to controls.

Gestation Length.There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

Litter Responses.

Offspring Litter Size and Viability.There were no treatment-related effects on litter size or offspring viability.

Offspring Growth and Development.Offspring bodyweight gain was slightly reduced from litters treated with 1000 mg/kg/day between Days 1 and 4post partum.

Pathology.

Necropsy. No toxicologically significant macroscopic abnormalities were detected for adults or offspring.

Organ Weights.Non-recovery animals of either sex treated with 1000 mg/kg/day showed an increase relative liver weight. Absolute liver weight was also elevated in non-recovery 1000 mg/kg/day females. No such effects were detected in animals of either sex treated with 250 or 50 mg/kg/day.

Histopathology.The following treatment-related changes were detected:

ADRENAL GLAND:Hypertrophy of the zona glomerulosa was seen in relation to treatment for males only at all treatment levels. This change is regarded as adaptive in nature and may be associated with renal changes.

The condition was observed to have regressed among Recovery 1000 mg/kg/day animals following an additional 14 days without treatment.

LIVER:Centrilobular hepatocyte enlargement was seen as a consequence of treatment for males treated with 1000 mg/kg/day but not at any other treatment level. There was no evidence of a similar effect for females.

The condition was observed to have regressed following completion of the recovery period.

Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive in nature.

KIDNEYS:Renal changes characterised by globular accumulations of eosinophilic material, tubular basophilia/degeneration, and tubular necrosis, were seen among males only treated with 1000 mg/kg/day. Globular accumulations of eosinophilic material were also seen for males treated with 250 mg/kg/day or at 50 mg/kg/day with tubular necrosis in a few males treated with 250 mg/kg/day. Similar accumulations were also demonstrated by examination of Mallory’s Heidenhain stained sections of kidney which assists in the diagnosis of but which is not diagnostic forα₂-Microglobulin.

There was regression of globular accumulations of eosinophilic material among Recovery 1000 mg/kg/day males following completion of the 14-day recovery period although residual tubular necrosis remained in four animals, possibly with an associated higher incidence/severity of isolated groups of basophilic tubules. 

The presence of globular accumulations of eosinophilic material is consistentwith hydrocarbon nephropathy, which results from the excessive accumulation ofα₂-microglobulinin renal proximal tubular epithelial cells. α₂-Microglobulin is found only in the proximal tubular epithelium of adult males.

THYROID:Follicular cell hypertrophy was seen in relation to treatment for males treated with 1000 mg/kg/day, but not convincingly at any other treatment level. Females were not similarly affected. There was no difference in incidence or severity of the condition between Recovery 1000 mg/kg/day and Recovery control animals following completion of the recovery period.

THYMUS:Lymphoid atrophy was observed as a consequence of treatment for females treated with 1000 mg/kg/day but not at any other treatment level. Males were not similarly affected.

The condition was observed to have regressed among recovery group animals following completion of an additional fourteen days without treatment.

Conclusion.The oral administration of Hatcol 1760 to rats by gavage, at dose levels of 50, 250 and 1000 mg/kg/day, resulted in treatment-related effect in males from all treatment groups and in females treated with 1000 mg/kg/day. A No Observed Effect Level (NOEL) has, therefore not been established for males however, no such changes were demonstrated at 250 mg/kg/day for females and the ‘No Observed Effect Level’ (NOEL) for females was, therefore considered to be 250 mg/kg/day.

The kidney changes identified histopathologically at 250 and 50 mg/kg/day were consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons. There is no evidence to suggest that the tubular necrosis seen in a few 250 mg/kg/day males is of independent origin since it is only seen in association with hydrocarbon nephropathy and was not observed among female rats. It is therefore considered to be associated with, or a consequence of, excessiveaccumulation of alpha-2-microglobulin in male rats in this investigation. This effect is, therefore, not indicative of a hazard to human health and, for the purposes of hazard evaluation, the “No Observed Adverse Effect Level” (NOAEL) for males, should be regarded as 250 mg/kg/day.

Offspring from litters treated with 1000 mg/kg/day showed a reduction in bodyweight gain between days 1 and 4post partum. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was therefore considered to be 250 mg/kg/day.

The effects seen at 1000 mg/kg/day are minimal and because of higher litter size and the fact that there is offspring bodyweight gain albeit at a lower rate than controls, the NOAEL can be suggested as 1000 mg/kg/day.