Petrolatum (petroleum), oxidized, Bu ester

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:

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.

Qualifier:

according to guideline

Guideline:

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

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

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

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

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.

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.

Duration of treatment / exposure:

Up to 54 days (males were sacrificed on Day 43; females were sacrificed on Day 5 post partum)

Frequency of treatment:

daily

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.

Parental animals: Observations and 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: males were weighed on day 0, then weekly until termination. 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
- Weekly food consumption was recorded for each caged group of adults, which was continued for males after mating. 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 lie 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.

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.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43.
- 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, epididymides, heart, kidneys, liver, ovaries, spleen, testes 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, epididymides*, eyes, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), mammary gland, muscle (skeletal), ovaries*, pancreas, pituitary*, prostate*, oesophagus, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles*, skin (hind limb), spinal cord (cervical), spleen, stomach, thyroidparathyroid, trachea, testes*, 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.

Postmortem examinations (offspring):

GROSS NECROPSY
All surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. They were subjected to a full external and internal examination, 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)

Reproductive indices:

MATING PERFORMANCE AND FERTILITY

The following parameters were calculated from the individual data during the mating period of the parental generation.

-Pre-coital interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

-Fertility Indices, for each group the following were calculated:
Mating Index (%) = [No. of animal mated/No. of animals paired] x 100
Pregnancy Index (%) = [No. of pregnant females/ No. of animals mated] x 100

GESTATION AND PARTURATION DATA

The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.

-Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition. Where the start of parturition occurred overnight, the total was adjusted by subtracting half a day.

-Parturition Index, the following was calculated for each group:
Parturition Index (%) = [No. of females delivering live offspring/No. of pregnant females] x 100

LACTATION DATA

The following indices were calculated for each group from individual data:

Live Birth Index (%) = [No. of offspring alive on day 1/No. of offspring born] x 100

-Sex ratio (% males), group mean values calculated from each litter value on day 1 and 4 using the following formula:
Sex ratio = [No. of male offspring/No. of offspring of determined sex] x 100

-Implantation losses (%), group mean percentile pre-implantation and post implantation loss were calculated as follows:
% pre-implantation loss = [(No. of Corpora Lutea – No. of implantation sites)/No. of corpora lutea] x 100
% post-implantation loss = [(Group no. of implantation sites – No. of offspring)/No. of implantation sites] x 100

Offspring viability indices:

Viability indices were calculated, as follows:

Viability Index (%) = [No. of offspring alive on day 4/No.of offspring alive on day 1] x 100

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

MORTALITY:
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 of either sex 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 male and one female treated with 1000 mg/kg/day during the Week 1 assessments. One 1000 mg/kg/day male also showed noisy respiration during Week 6.

BODYWEIGHT:
There were no adverse effects on bodyweights for males throughout the study. 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. The statistically significant reductions in dietary intake detected for all male treatment groups during the final week of treatment were minimal (p<0.05) and attributable to a slightly higher dietary intake for the controls and considered unrelated to treatment.

REPRODUCTIVE PERFORMANCE:
No adverse effects were detected on mating performance or fertility. One female treated with 1000 mg/kg/day showed positive evidence of mating after one day of pairing, however, this animal failed to achieve pregnancy. This is a normal low incidence finding on reproductive studies of this type and in isolation, considered unrelated to treatment. A reduced parturition index was evident at 1000 mg/kg/day, but this was attributed to the termination of one female due to dystocia. There were no treatment-related intergroup differences in litter sizes, sex ratio or live birth indices.

ORGAN WEIGHTS:
No toxicologically significant effects of treatment were detected.
Elevated liver weights, relative to terminal bodyweights were detected for animals (of either sex treated with 1000 mg/kg/day, with statistical significance achieved for males (p < 0.01). 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. The statistically significant reduction in absolute adrenal and heart weights detected for males treated with 1000 mg/kg/day were not reflected in the relative weights and therefore considered unrelated to treatment. The statistically significant increase in relative liver weight detected for 100 mg/kg/day males was minimal (p < 0.05) and in the absence of a convincing dose related response, was considered incidental and unrelated to treatment.

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. One male showed dorsal scab formation at termination but this isolated incident was unrelated to treatment.
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:
- Kidneys: Globular accumulations of eosinophilic material were observed in the tubular epithelium of males treated at all treatment levels. One control male was similarly affected. There was no indication of associated degenerative tubular changes and no dose relationship. This finding is consistent with the presence of hydrocarbon nephropathy following treatment with petroleum based test materials. This results from the excessive accumulation of alpha2-microglobulin in renal proximal tubular epithelial cells, found only in the proximal tubular epithelium of adult male rats.
- Thyroids: A marginal effect was observed in respect of the prevalence and severity of follicular cell hypertrophy in relation to treatment for animals of either sex treated with 1000 mg/kg/day. A similar effect was also seen for males treated with 300 mg/kg/day, extending into the 100 mg/kg/day dose group. Although not convincing due to similar findings in the controls, the possibility that treatment with the test material has 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 two male rats and 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.

Dose descriptor:

NOAEL

Remarks:

(Reproduction)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY:
- There were no treatment-related intergroup differences in post partum viability.
The interim deaths of offspring from the control, 100 and 300 mg/kg/day treatment groups during the lactation phase of the study are normal low incidence findings and considered unrelated to treatment.

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.

Reproductive effects observed:

not specified

Results of Preliminary toxicity Study:

- Increased salivation was detected up to ten minutes after dosing for animals of either sex treated with 1000 mg/kg/day from Day 2 onwards. This observation is often recorded following the oral administration of an unpleasant tasting and/or locally irritant test material formulation and in isolation, is not considered to be indicative of systemic toxicity.

- No mortalities were observed or systemic signs of toxicity.

Conclusions:

Under the conditions of the study there was no evidence of reproductive toxicity relating to treatment 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. Under the conditions of the study there was no evidence of reproductive toxicity relating to treatment with the test material. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was therefore considered to be 1000 mg/kg bw/day.

Executive summary:

In a GLP compliant study the reproductive 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. Examination of reproductive performance offspring litter sizes, sex ratios, reproductive and viability indices showed that there was no evidence of reproductive toxicity during the course of 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. Under the conditions of the study there was no evidence of reproductive toxicity relating to treatment with the test material. The ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive toxicity was therefore considered to be 1000 mg/kg bw/day.

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:

The study was performed in line with GLP and an accepted standardised guideline with a high standard of reporting. The study was performed with a structural analogue and so was assigned a reliability score of 2 in accordance with the criteria for assessing data quality as outlined in Klimisch (1997) and considered suitable for assessment as an accurate reflection of the test material. The oral route was considered the most appropriate route.

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 study the reproductive 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. Examination of reproductive performance offspring litter sizes, sex ratios, reproductive and viability indices showed that there was no evidence of reproductive toxicity during the course of 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. Under the conditions of the study there was no evidence of reproductive toxicity relating to treatment with the test material. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was therefore considered to be 1000 mg/kg bw/day.

 

Since the study was performed with a structural analogue it was assigned a reliability score of 2 in accordance with the criteria for assessing data quality as outlined in Klimisch (1997). The results of this read-across study can be considered to represent a worst case since the test material (Distillates (petroleum), oxidized light) is a shorter chain material and therefore has a greater potential for absorption within the body. The read-across substance can therefore be considered to be potentially more bioavailable than the registered substance, which, due to its very high log Pow value, is anticipated to have only very limited potential for bioavailability.

 

In accordance with Column 2 (adaptation statement) of Annex IX of Regulation (EC) 1907/2006 (REACH), it is considered justified to omit the two-generation reproductive toxicity study required under information point 8.7.3 if the substance is of low toxicological concern. The toxicological activity of the substance is negligible and the toxicokinetic assessment of the registered substance shows low potential for absorption.

In a reliable, well reported combined repeated dose oral toxicity and reproduction/developmental screening study in rats, conducted with a structural analogue, the NOAEL was determined as 1000 mg/kg bw/day (actual dose received) for reproduction effects.

Short description of key information:
ORAL
NOAEL (Reproductive toxicity) = 1000 mg/kg bw/day (distillates (pertoleum), oxidized light), rat (male/female), OECD 422, Dhinsa (2005)

Justification for selection of Effect on fertility via oral route:
Only one study is available.

Effects on developmental toxicity

Description of key information

ORAL
NOAEL (maternal toxicity) = 1000 mg/kg bw/day; NOAEL (developmental toxicity) = 1000 mg/kg bw/day (distillates (pertoleum), oxidized light), rat (male/female), OECD 422, Dhinsa (2005)

Link to relevant study records

Reference

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.

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

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

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.

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.

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.

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.

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

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

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.

Abnormalities:

not specified

Developmental effects observed:

not specified

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.

Effect on developmental toxicity: 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:

The study was performed in line with GLP and an accepted standardised guideline with a high standard of reporting. The study was performed with a structural analogue and so was assigned a reliability score of 2 in accordance with the criteria for assessing data quality as outlined in Klimisch (1997) and considered suitable for assessment as an accurate reflection of the test material. The oral route was considered the most appropriate route.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

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/day.

 

Since the study was performed with a structural analogue it was assigned a reliability score of 2 in accordance with the criteria for assessing data quality as outlined in Klimisch (1997). The results of this read-across study can be considered to represent a worst case since the test material (Distillates (petroleum), oxidized light) is a shorter chain material and therefore has a greater potential for absorption within the body. The read-across substance can therefore be considered to be potentially more bioavailable than the registered substance, which, due to its very high log Pow value, is anticipated to have only very limited potential for bioavailability.

 

In accordance with Column 2 (adaptation statement) of Annex IX of Regulation (EC) 1907/2006 (REACH), it is considered justified to omit the pre-natal toxicity study required under information point 8.7.3 if the substance is of low toxicological concern. The toxicological activity of the substance is negligible and the toxicokinetic assessment of the registered substance shows low potential for absorption.

In a reliable, well reported combined repeated dose oral toxicity and reproduction/developmental screening study in rats with a structural analogue, the NOAEL was determined to be 1000 mg/kg bw/day (actual dose received) for developmental effects. The lack of toxicity observed at such a high dose level indicates that performing a longer term study is scientifically unnecessary and inappropriate on animal welfare grounds.

Justification for selection of Effect on developmental toxicity: via oral route:
Only one study is available.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation 1272/2008 (CLP) and Directive 67/548/EEC (DSD)

, the test material does not require classification for reproductive or developmental toxicity.