N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 1992 - March 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

EC Number:

204-029-1

EC Name:

N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

Cas Number:

113-48-4

Molecular formula:

C17H25NO2

IUPAC Name:

N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

impurity 1

Reference substance name:

(E)-1,4-Bis(2-ethylhexylamino)-2-butene-1,4-dione

Molecular formula:

C20H38N2O2

IUPAC Name:

(E)-1,4-Bis(2-ethylhexylamino)-2-butene-1,4-dione

impurity 2

Reference substance name:

1-(2-ethylhexyl)-1H-pyrrole-2,5-dione

Molecular formula:

C12H19NO2

IUPAC Name:

1-(2-ethylhexyl)-1H-pyrrole-2,5-dione

Test material form:

liquid

Details on test material:

Purity and characterisation analysis conducted on the following sample; Supplier: McLaughlin Gormley King ; Batch Number: AB9500

Specific details on test material used for the study:

The test article; a pale yellow liquid, used for this study came from a 17kg (gross weight) sample received at Toxicol on 16th September 1991. It was supplied in a grey metal drum as MGK 264 Synergist and labelled as follows : Lot 3843; No 1 of 1; G 38.19; T 4.5; N 33.69
When not in use the test article was stored at room temperature in the dark .

Test animals

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

Forty-five male and 45 female New Zealand 2.5 to 3.1 kg in weight on arrival, were were supplied by Interfauna U.K. Ltd.,
England. White rabbits, 11-17 weeks old and delivered on 6th March 1992. All animals were found to be healthy on arrival. During the acclimatisation period 2 animals were lethargic and were euthanased. The animals were acclimatised for 18 days instead of the 7-14 days required by the protocol. The surviving animals were re-examined, confirmed to be suitable for experimental use and 80 were selected for the study. Predose ophthalmoscopy detected an ocular defect in one male animal. This animal was rejected from the study and replaced with a male, from the same batch. At the start of treatment the males weighed 3.0 - 3.6kg and the females weighed 2.9 - 3.6kg.

The animals were individually housed in grid-bottomed metal cages (Biotech/Stephen Clark Fabrications) suspended over cardboard lined excreta trays. The experimental rooms (Bl and 2) were illuminated by fluorescent lighting controlled automatically to give a cycle of 12 hours light (0600 to 1800 hours), and 12 hours dark. They were air conditioned and recorded temperature and relative humidity were in the ranges 16 - 25°C and 41 - 72% respectively. The temperature in room Bl rose above the protocol specified upper limit (21°C) by 1°C on five occasions, and by 2°C on three occasions, (see deviations, page 7). The high relative humidity of 72% was recorded on two occasions in this room. In room B2, the temperature rose above the protocol specified upper limit by 1°C on sixteen occasions, by 2°C on three occasions, by 3°C on one occasion and by 4°C on one occasion.

All animals were offered a pelleted, antibiotic-free, rabbit diet (SQC Stanrab (P) Special Diets Services Limited, Witham, England). Mains water was provided by an automatic watering system. With the exception of periods of deprivation associated with laboratory investigations (see section 4.3) both were freely available throughout the study.

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

corn oil

Details on exposure:

Twenty-four hours before the start of treatment, the application sites were clipped free of fur. During the treatment period the application sites were clipped as often as necessary to maintain them free of fur.
The test article was applied dermally to a clipped area (about 10 x 10 cm) of the upper back. Animals were dosed once daily, 7 days a week for 13 weeks The application site was then covered with a semi-occlusive dressing for a period of 6 hours. On day 82 only, the test article applied to animal 52 (group 2F) was left in contact with the back for approximately 24 hours because of a technical oversight. When this was discovered on day 83, the back was immediately washed. The animal was allowed a period of rest and was then dosed. The dressing consisted of a piece of gauze placed over the application site and a elastic stockinette tubular bandage (12cm diameter, supplied by IMS Limited, Cheshire, U.K.). Holes were then made in the tubular bandage for the legs, to prevent the dressing from slipping. At the end of each exposure period, the application site was thoroughly washed with warm.water and a mild soap ('Simple', ex. Smith and Nephew Ltd., Birmingham, England) to remove as much residual test article as possible and the skin was then blotted dry with a paper towel. Elizabethan collars were fitted for the duration of each 6-hour exposure period, to prevent oral ingestion of the test article.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

As a check on the accuracy of preparation, samples of each formulation (including the control formulation) prepared on one day in each of weeks 1 and 13 were analysed for test article at Toxicol Lahoratories. (For procedural details and results refer to Appendix 13 in the final report attached to this end point study record).

Duration of treatment / exposure:

The test article was applied dermally to a clipped area (about 10 x 10 cm) of the upper back. Animals were dosed once daily, 7 days a week for 13 weeks. Elizabethan collars were fitted for the duration of each 6-hour exposure period, to prevent oral ingestion of the test article.

Frequency of treatment:

Animals were dosed once daily, 7 days a week for 13 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

controls - recieived the vehicle (corn oil).

No. of animals per sex per dose:

10 male / 10 female per dose. A constant dose volume of 1 ml/kg bodyweight was used.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected in light of previous 7 and 14 day range finding studies.
In the 7 day preliminary range-finding study with MGK 264, doses of neat material as high as 1000 mg/kg/day were used and were associated with severe skin reactions.
For a subsequent 14 day preliminary range-finding study MGK 264 was diluted in corn oil and doses up to l100 mg/kg/day were used. Due to the degree of skin irritation noted at 30 and 100 mg/kg/day, in this study and since the definitive GLP study was to be of 90 days duration, it was considered that if such irritation was allowed to continue, possibly becoming more pronounced, any debility could be due to the irritation and not due to systemic toxicity. Therefore, it was decided that a dose level of MGK 264 in corn oil not exceeding 100 mg/kg/day should be used in order to avoid severe chronic skin reactions of animals on the study, especially as 7 days/week dosing was planned.

Examinations

Observations and examinations performed and frequency:

During the treatment period, bodyweights and food consumption were measured weekly and skin reactions and clinical observations were recorded daily.
Ophthalmological examinations were carried out on all animals before treatment started and on control and high dose animals during week 13 of the study. Blood samples were taken for haematology and blood chemistry in week 13 of the study.

Sacrifice and pathology:

At the end of the treatment period, all animals were killed, necropsied and the weights of the kidneys, liver and testes were recorded. A range of tissues was preserved for subsequent histopathology .

Statistics:

Bodyweight, haematological and organ weight data were evaluated by analysis of variance (ANOVA) and, if a between groups difference significant at the 5% level occurred, by pairwise t-tests between the control and treatment groups.
Blood chemistry data were analysed by Kruskal-Wallis ANOVA and, if a between group difference significant at the 5% level occurred, significant differences between the control and treatment group were determined by Multiple Comparison test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

Observations recorded during the study included: pustule formation, desquamation, eschar formation, wrinkling of the skin, erythema and oedema.
Erythema was seen to cover 10 - 100% of the appliration site in group 1, 20-100% in groups 2 and 4 and 10-100% in group 3. Oedema covered 10-100% of the application site in group 1, 20-100% in group 2, 10-100% in group 3 and 40-100% in group 4. Skin reaction scores for hnth erythema and oedema ranged from 0 (no erythema or oedema) to 3 (moderate to severe erythma or moderate oedema) in control and treated animals. Occasional scores of 4 (severe erythema or oedema) were seen in group 3 and 4 males.
Varying degrees of pustule formation, desquamation and eschar formation were seen in animals from all groups.
It was concluded that the skin reactions observed were due mainly to the semi-occlusive dressing and corn oil vehicle and were not related to treatment with MGK 264 Synergist.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related microscopic changes .
Minimal or moderate epidermal hyperplasia, with associated chronic inflammation in the dermis in many instances, was present in the treated skin of the majority of control and high dose group animals. Focal scab formation and minimal hyperkeratosis were seen in a few control and treated animals. Superficial haemorrhage was noted in one group 3 male and one group 3 female.
All other minor changes observed showed no group relationship and were recognised as that which can occur spontaneously in the laboratory rabbit.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Administration of MGK 264 dermally to the rabbit for 13 weeks was not associated with any MGK 264 related adverse effects in any of the treament groups.

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The administration of MGK 264 dermally to the rabit, daily for 13 weeks, at dose levels up to 100 mg/kg/day was not associated with any overt signs of toxicity .

Executive summary:

Eighty New Zealand White rabbits (40 males and 40 females) were divided into four groups of 10 males and 10 females. Three groups received a solution of MGK 264 in corn oil, dermally, at dose levels of 10, 30 or 100 mg/kg/day. The remaining group received the vehicle (corn oil) alone, at the same dose volume (1 ml/kg) and served as a control. Dose levels in this study were selected based on the findings of 7 day and 14 day dose range finding studies in rabbits in which skin irritation was seen at dermal applications of 30 mg/kg bw/day or more.

Animals were dosed once daily, 7 days a week for 13 weeks. The test article was left in contact with the skin for 6 hours a day under a semi-occlusive dressing. At the end of each exposure period, the backs were washed with soap and water.

During the treatment period, bodyweights and food consumption were measured weekly and skin reactions and clinical observations were recorded daily.

Ophthalmological examinations were carried out on all animals before treatment started and on control and high dose animals during week 13 of the study. Blood samples were taken for haematology and blood chemistry in week 13 of the study.

At the end of the treatment period, all animals were killed, necropsied and the weights of the kidneys, liver and testes were recorded. A range of tissues was preserved for subsequent histopathology.

No treatment related effects were noted during the study. Microscopic examination identified minimal or moderate epidermal hyperplasia, with associated chronic inflammation in the dermis in many instances, present in the treated skin of the majority of control and high dose group animals. Focal scab formation and minimal hyperkeratosis were seen in a few control and treated animals. None of these findings were related to treatment.

It was concluded that the administration of MGK 264 dermally to the rabbit, daily for 13 weeks, at dose levels up to 100 mg/kg/day was not associated with any overt signs of toxicity.