Petrolatum (petroleum), oxidized, Bu ester

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

4 August 2005 to 15 February 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

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. The study report was conclusive, conducted to a valid guideline and was performed under GLP conditions. Since the study was conducted with the read across substance, distillates (pertoleum), oxidized light, it has been assigned a reliability score of 2. Read across from this substance is justified on the basis of its similar physical and chemical properties to those of the registered substance. Furthermore, it has a very similar chemical structure; the registered substance is longer chain material; it is also an esterified form of the read across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

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)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Distillates (petroleum), oxidized light

EC Number:

265-203-0

EC Name:

Distillates (petroleum), oxidized light

Cas Number:

64742-98-9

IUPAC Name:

64742-98-9

Test material form:

liquid: viscous

Details on test material:

- Physical state: Brown slightly viscous liquid
- Storage conditions: Room temperature in the dark

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Sprague-Dawley Crl:CD® (SD) IGS BR
- Source: Charles River (UK) Limited, Margate, Kent, UK
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 201 - 266 g (males); 177 - 238 g (females)
- Housing: initially in groups of 4, during the mating phase animals were housed in pairs (one of each sex), following mating females where housed individually and males in groups. Cages were polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper. Mated females were housed in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes (Datesand Ltd. Cheshire, UK).
- Diet: pelleted diet (Rodent PMI 5002 (Certified) diet, BCM IPS Limited, London, UK), ad libitum
- Acclimation period: Up to 16 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The appropriate concentrations were prepared in the vehicle weekly and stored at approximately 4 °C in the dark.
- The test material was administered daily by gavage using a plastic catheter attached to a disposable plastic syringe.
- Control animals were treated in an identical manner with 4 mL/kg/day of the vehicle.
- The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at regular intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples from each preparation were analysed using gas chromatography to verify the concentration, stability and homogeneity of each formulation.

Method:
- Gas chromatography.
- Samples and standards were prepared in methanol to give a final concentration of 0.1 mg/mL
- Samples were analysed within two days of preparation
- The method was validated using a range of standard solutions covering the range 0 to 0.1527 mg/mL. The results of which have been considered to be sufficiently accurate for the purpose of this study.

Conditions:
- GC system: Agilent Technologies 5890, incorporating autosampler and workstation
- Column: DB-1 (30 m x 0.32 mm id x 0.25 µm film)
- Oven temperature program: initial 50 °C for 0 mins, rate 5 °C/min, final 200 °C for 0 mins
- Injection temperature: 150 °C
- Flame ionisation detector temperature: 250 °C
- Injection volume: 1 µL
- Retention time: ~14.7 mins

Results:
- The results indicate that the prepared formulations were within ± 9 % of the nominal concentration.
- Analytical verification of the stability and homogeneity of the test preparations was also performed and the formulation was found to be stable for at least fourteen days.

Details on mating procedure:

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 and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Duration of treatment / exposure:

Up to 54 days

Frequency of treatment:

Daily

Duration of test:

Males were sacrificed on Day 43; females were sacrificed on Day 5 post partum. Offspring were sacrificed on Day 5 post partum.

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10 males and 10 females per dose level

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: The animals were allocated to dose groups using a randomisation procedure based on stratified bodyweights and the group mean bodyweights were then determined to ensure similarity between the dose groups.

- Dose selection rationale:
- A fourteen day oral range finding study was conducted to provide information for the selection of dose levels for the definitive study. Animals were dosed and observed for adverse effects resulting from toxicity including; clinical observations, bodyweight and gross pathology.
- Animals were kept under the same conditions as in the definitive study.
- The test material was prepared in the same manner as the definitive study and verified analytically.
- The test material was administered in a limit test daily, for up to 14 consecutive days. Animals were administered 1000 mg/kg/day via oral gavage, a volume of 4mL/kg and a concentration of 250 mg/mL. Control animals were treated in an identical manner with 4 mL/kg/day of polyethylene glycol.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- 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: date of mating, date and time of observed start of parturition, date and time of observed completion of parturition and duration of gestation.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before and after dosing, then at 1 and 5 hours after dosing. The 5 hour observation was omitted at weekends, except for females during parturition where applicable.
- Observations recorded: overt signs of toxicity, ill-health and behavioural changes.

BODY WEIGHT: Yes
- Time schedule for examinations: Females were weighed on day 0 and then weekly until evidence of mating was evident. Bodyweights were recorded on day 0, 7, 14 and 20 post coitum, and on day 1 and 4 post partum.

FOOD AND WATER CONSUMPTION: Yes
- Food consumption for females showing evidence of mating was recorded for the periods covering days 1-7, 7-14 and 14-21. For females with live litters, consumption was recorded during the lactation period.
- Water consumption was observed daily by visual inspection of water bottles for any overt change.

OTHER:
As part of the repeated dose toxicity assessment of the test material, functional observational battery tests were conducted together with behavioural assessments and neurobehavioural examinations. Furthermore, haematology and clinical chemistry investigations were conducted.

POST-MORTEM EAXMINATIONS
SACRIFICE
- Maternal animals: The interim death female was killed by carbon dioxide asphyxiation followed by cervical dislocation. The remaining surviving adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum.

GROSS NECROPSY
- All adult animals, including those dying during the study, were subjected to 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. The procedure was enhanced by staining the uteri with a 1 % ammonium polysulphide solution.

ORGAN WEIGHTS
- The following organs were dissected free from fat and weighed at the end of the study; adrenals, brain, heart, kidneys, liver, ovaries, spleen, and the thymus.

HISTOPATHOLOGY: Yes
Samples of the following tissues were preserved from five males and five females, from each dose group: adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, coagulating gland*, colon, duodenum, eyes, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), mammary gland, muscle (skeletal), ovaries*, pancreas, pituitary*, oesophagus, rectum, salivary glands (submaxillary), sciatic nerve, skin (hind limb), spinal cord (cervical), spleen, stomach, thyroidparathyroid, trachea, thymus, urinary bladder, uterus/cervix* and the vagina.
The tissues from five selected control and 1000 mg/kg/day dose group animals and those animals dying during the study, were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination. Tissues marked with an asterisk were also processed from the remaining control and 1000 mg/kg/day animals.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- The procedure was enhanced by staining the uteri with a 1 % ammonium polysulphide solution.

Fetal examinations:

LITTER OBSERVATIONS
- On completion of parturition, the number of live and dead offspring was recorded.
- For each litter the following was recorded: number of pups born, number and sex of pups alive recorded daily and reported on Day 1 and 4 post partum, clinical condition of pups from birth to Day 4 post partum, individual pup and litter weights on Day 1 and 4 post partum.
- All live offspring were observed for the detachment of pinna and assessed for reflexological response to stimuli by assessing surface righting reflex on Day 1 post partum.

POST-MORTEM EXAMINATIONS
No pre-parturition examinations were performed. All offspring, including those dying during the study, were subjected to a full external and internal examination post parturition, and any macroscopic abnormalities were recorded.

Statistics:

Data were processed to give group mean values and standard deviations where appropriate. Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain 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 haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value.

Probability values (p) are presented as follows:
p < 0.001 ***
p < 0.01 **
p < 0.05 *
p 20.05 (not significant)

Indices:

Mating index, fertility index, parturition index and viability index were calculated.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY:
No treatment related deaths were observed during the course of the study. One female treated with 1000 mg/kg/day was killed in extremis during parturition. This was attributed to dystocia and of no toxicological significance.

CLINICAL OBSERVATIONS:
Increased salivation was detected prior to dosing and up to one hour after dosing for animals treated with 1000 mg/kg/day from Day 3 onwards. An isolated incident of increased salivation was also detected up to one hour after dosing for one female treated with 300 mg/kg/day on Day 17 only. This observation is often recorded following the oral administration of an unpalatable or irritant test material. Isolated incidents of noisy respiration were also observed at 1000 mg/kg/day.
No such effects were detected at 100 mg/kg/day. Incidents of scab formation, generalised fur loss and staining of the external body surface were observed throughout the treatment and control groups. These are occasionally seen in laboratory maintained rodents and are considered to be incidental and not indicative of toxicity.
Isolated incidents of noisy respiration were detected for one female treated with 1000 mg/kg/day during the Week 1 assessments.

BODYWEIGHT:
Bodyweight gains for treated females throughout the maturation, gestation and lactation phases of the study were comparable to controls.

FOOD COMSUMPTION:
No adverse effects were observed on food consumption during the course of the study.

REPRODUCTIVE INDICES:
No treatment related effect on copulation index, fertility index, delivery index and viability index was observed when compared with controls.

ORGAN WEIGHTS:
No toxicologically significant effects of treatment were detected.
Elevated liver weights, relative to terminal bodyweights were detected for animals treated with 1000 mg/kg/day. Elevated adrenal weights, both absolute and relative to terminal bodyweight was detected for females treated with 1000 mg/kg/day. In the absence of histopathological correlates, these effects were most probably attributed to xenobiotic induction and of no toxicological importance.

NECROPSY/GROSS PATHOLOGY:
The interim death pregnant female treated with 1000 mg/kg/day showed four placentae and four dead foetuses in the right uterine horn. The left uterine horn was blood filled. This animal was terminated due to difficulties in birthing. Dystocia is a common low incidence finding in reproductive studies of this type and in isolation, this death was considered unrelated to test material toxicity. No treatment-related macroscopic abnormalities were detected for adult animals at terminal kill.
No treatment-related changes were detected in corpora lutea and implantation site counts. Statistical analysis of the data did not reveal any significant differences.

HISTOPATHOLOGY
The following treatment-related changes were observed:
- Thyroids: A marginal effect was observed in respect of the prevalence and severity of follicular cell hypertrophy in relation to treatment for animals treated with 1000 mg/kg/day. Although not convincing due to similar findings in the controls, the possibility that treatment with the test material had affected the thyroid follicular epithelium cannot be entirely excluded; this can, however, be generally regarded as an adaptive change.
- Stomach: Minimal acanthosis, occasionally with associated hyperkeratosis, was observed in relation to treatment for three females treated with 1000 mg/kg/day, but not at any other treatment level. These are adaptive responses, secondary to oral gavage administration of a highly concentrated epithelial irritant.

All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
VIABILITY:
- There were no treatment-related intergroup differences in post partum viability.

CLINICAL SIGNS:
- No toxicologically significant clinical findings were observed. The clinical observations recorded for offspring throughout the dose groups were consistent with normally expected incidence findings in offspring of the age examined and were of no toxicological importance. Surface righting or pinna unfolding assessments for treated offspring were comparable to controls.

BODY WEIGHT:
- There were no significant intergroup differences in mean litter weights or offspring bodyweights.

GROSS PATHOLOGY:
- No treatment-related macroscopic abnormalities were detected for offspring at terminal kill. The macroscopic findings observed for interim and terminal kill offspring throughout the dose groups, were consistent with normally expected low incidence findings in pups of the age examined and were of no toxicological importance.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study there was no evidence of maternal toxicity or developmental toxicity following treatment of parental animals with the test material. One high dose female failed to achieve pregnancy and another female from this dose group was sacrificed at parturition due to dystocia. These are normal low incidental findings observed on reproductive studies and considered unrelated to treatment. No adverse effects were recorded in offspring growth or development, therefore the ‘No Observed Effect Level’ (NOEL) for developmental toxicity was considered to be 1000 mg/kg bw/day.

Executive summary:

In a GLP compliant study the developmental toxicity of the test material was determined in a combined repeat dose toxicity study with reproduction/developmental toxicity screening test in the rat. The study was performed according to the standardised guideline OECD 422. During the study groups of 10 male and 10 female Sprague-Dawley rats were administered test material daily, at concentrations of 100, 300 or 1000 mg/kg, for a period of up to 54 days. Clinical signs, bodyweight development, food and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male to 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. Males were terminated on Day 43 while females were terminated on Day 5 post partum. All animals were subjected to a gross necropsy examination and histological evaluation of selected tissues, from the control and 1000 mg/kg dose group animals was performed.

No adverse effects or systemic signs of toxicity were recorded as a result of treatment during this study. One high dose female failed to achieve pregnancy and another female from this dose group was sacrificed at parturition due to dystocia. These are normal low incidental findings observed on reproductive studies and considered unrelated to treatment. No treatment related effects were detected in offspring growth or development, therefore the ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity was considered to be 1000 mg/kg bw/day.