Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1988-05-16 to 1990-02-12
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study was conducted in methods comparable to OECD guideline 411 "Subchronic Dermal Toxicity: 90-day study" and under GLP. However, there were only 4-6 males and females at each dose level. High dose group was terminated after Week 3 due to severe skin irritation. For Read across justification refer to IUCLID section 13 Assessment reports.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
See rationale for reliability above.
Principles of method if other than guideline:
N/A
GLP compliance:
no
Remarks:
A QAU inspection certificate was included.
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): E-3333.01
- Chemical name: C13-15 dimethyl hydroxyethylammonium chloride
- Molecular formula (if other than submission substance): N/A
- Molecular weight (if other than submission substance): N/A
- Smiles notation (if other than submission substance): N/A
- InChl (if other than submission substance): N/A
- Structural formula attached as image file (if other than submission substance): N/A
- Analytical purity: 28.15 % active
- Physical state: clear liquid
- Purity test date: 1987-09-30
- Lot/batch No.: N/A
- Expiration date of the lot/batch: September 1988
- Isomers composition: N/A
- Other: Storage-room temperature/ protect from light

Test animals

Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Rabbits were from HUK stock that were previously obtained from Hazleton Research Products, USA
- Age at study initiation: N/A
- Weight at study initiation: 2.0-3.0 kg (however, six males and two females were heavier than 3.0 kg at start of dosing)
- Fasting period before study: N/A
- Housing: housed singly on grid floors
- Diet: ad libitum up to 125g/day of Standard Rabbit Diet, analyzed for specific contaminants.
- Water: ad libitum
- Acclimation period: minimum of 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 12-24°C
- Humidity (%): 40-86%
- Air changes (per hr): minimum 10/hr
- Photoperiod (hrs dark / hrs light): 10 hours dark/14 hours light

IN-LIFE DATES: From: N/A To: N/A

Administration / exposure

Type of coverage:
not specified
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: Approximately 10 X 10 cm
- % coverage: N/A
- Type of wrap if used: N/A
- Time intervals for shavings or clippings: as needed


REMOVAL OF TEST SUBSTANCE
- Washing (if done): N/A
- Time after start of exposure: N/A


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2mL/kg (weeks 1 and 2), 5mL/animal (from week 3 on)
- Concentration (if solution): 0, 0.02%, 0.2 %, 2% w/v
- Constant volume or concentration used: no
- For solids, paste formed: N/A


VEHICLE
- Justification for use and choice of vehicle (if other than water): distilled water
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples (10ml) were taken from formulations prepared for study week 1 (0, 0.2 % w/v, and 2.0 % w/v groups), week 3 (residues from 0 and 0.2 % w/v groups, and formulation from 0.02% w/v group- dosing week 1), week 13 (0 and 0.2 % w/v groups) and week 14 (0.02% w/v group- dosing week 12). The samples were stored deep-frozen before analysis.
Duration of treatment / exposure:
13 weeks (0 and 0.2 % w/v groups), 2 weeks (2.0 % w/v group), and 12 weeks (0.02% w/v group)
Frequency of treatment:
daily, 5 days a week
Doses / concentrations
Remarks:
Doses / Concentrations:
0.0% (control), 0.02% w/v, 0.2% w/v, and 2.0% w/v
Basis:
other: the test concentrations were verified to be 100 to 103% of nominal concentrations, 2ml/kg were administered the first two weeks and 5ml/animal were administered from week 3
No. of animals per sex per dose:
5/sex/dose, except in 0.02% group which had 4 males and 6 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous dose range-finding study
- Rationale for animal assignment (if not random): randomization based on stratified body weight during the acclimatization period
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random): N/A
Positive control:
N/A

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table, all animals were included for ill-health or overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily (24 hours after dosing)

BODY WEIGHT: Yes
- Time schedule for examinations: initially, and then weekly

FOOD CONSUMPTION: N/A
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: N/A

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in week 13 (0 and 0.2% w/v groups) and in week 12 (0.02% w/v group)
- Anesthetic used for blood collection: No data
- Animals fasted: Yes, for 18 hours
- How many animals: all except for those in the 2.0 % w/v dose group
- Parameters checked in table [No.?] were examined. N/A

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in week 13 (0 and 0.2% w/v groups) and in week 12 (0.02% w/v group)
- Animals fasted: Yes, for 18 hours
- How many animals: all except for those in the 2.0 % w/v dose group
- Parameters checked in table [No.?] were examined. N/A

URINALYSIS: No data
- Time schedule for collection of urine: N/A
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. N/A

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A
- Battery of functions tested: N/A

OTHER: N/A
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, organ weights before fixation were done on the adrenals, kidneys, lungs, testes, heart, liver, and ovaries. Also, “a full external and internal examination was made in the presence of the study pathologist and all gross lesions were recorded”.

HISTOPATHOLOGY: Yes: adrenals, caecum, duodenum, eyes, gall bladder, heart, jejunum, liver, lymph nodes, ovaries, pituitary, rectum, sciatic nerve, seminal vesicles, spinal cord, stomach, thymus, untreated skin, uterus, vagina, aorta, brain, colon, epididymides, ileum, kidneys, lungs, mammary glands, esophagus, pancreas, prostate, salivary glands, skeletal muscle, spleen, sternum, testes, thyroids, treated skin, ureters, urethra, urinary bladder, all gross lesions.

Bone marrow smears were made but not evaluated as there were no haeematological parameters.
The trachea, femur and sternebrae were retained in fixative and not processed further.

Other examinations:
N/A
Statistics:
Data were processed, where appropriate, to give group mean values and standard deviations. No further statistical analyses were performed.

Results and discussion

Results of examinations

Clinical signs:
not examined
Description (incidence and severity):
Animals in high dose group (2 % w/v) killed for humanitarian reasons due to severity of skin reactions
Dermal irritation:
effects observed, treatment-related
Mortality:
not examined
Description (incidence):
Animals in high dose group (2 % w/v) killed for humanitarian reasons due to severity of skin reactions
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
High dose group only, secondary effect due to irritation
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
local skin effects in high dose group
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
only local findings on the skin
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY: All animals in the 2.0% w/v group were killed and necropsied in week three owing to the severity of the skin reactions. During weeks 1 and 2 several animals from this dose group were noted to be not eating and in weeks 2 and 3 several animals were thin. No clinical signs were observed in any of the other dose groups.

BODY WEIGHT AND WEIGHT GAIN: All animals in the 2.0% w/v group, except one, lost weight over the period they were dosed. During week 2, 0.2% male animals lost weight. From week 3, the body weight gains of the males in this group and the control group were comparable. Females’ mean body weights from the 0.2% w/v group were similar to those of the control group’s throughout the study. Group mean body weights of the 0.02% w/v group (males and females) were similar to those of the control.

FOOD CONSUMPTION: N/A

FOOD EFFICIENCY: N/A

WATER CONSUMPTION: N/A

OPHTHALMOSCOPIC EXAMINATION: N/A

HAEMATOLOGY: The animals in the 0.2% w/v had mean white blood cell counts less than controls by 14% and 11% for males and females, respectively. However, all individual values were within normal ranges and therefore, this is considered to be of no toxicological significance. There were no apparent effects on other hematology parameters.

CLINICAL CHEMISTRY: There was no apparent effect of treatment on clinical chemistry parameters.

URINALYSIS: N/A

NEUROBEHAVIOUR: N/A

ORGAN WEIGHTS: The group mean relative and absolute, lung and heart, weights of the 0.02% w/v males were greater than those of the control group. However, there was no apparent effect in the females or in the 0.2% w/v animals, thus no dose relationship and therefore it is considered to be of no toxicological significance.

GROSS PATHOLOGY: No significant effects were seen in either the control animals or animals dosed with 0.02% w/v. In the 0.2% and 2.0% w/v groups there was a dose-related dermatitis. At necropsy this appeared as thickening of the skin with fissuring and, in 2.0% group animals, was accompanied by sloughing of the superficial layers.

HISTOPATHOLOGY: NON-NEOPLASTIC: No significant effects were seen in either the control animals or animals dosed with 0.02% w/v. In the 0.2% w/v group there was dermatitis and in the 2.0% w/v group, incomplete re-epithelialization of the tissue underlying the sloughed epidermal layers. Also, there was minor hyaline droplet accumulation in the kidney of one female from the 0.2% group and all animals in the 2.0% w/v group. The hyaline droplet formation was identified as being a secondary effect to the skin irritation rather than a direct toxic effect.

HISTOPATHOLOGY: NEOPLASTIC (if applicable): N/A

HISTORICAL CONTROL DATA (if applicable): N/A

OTHER FINDINGS: SKIN IRRITATION: All animals in the 2.0% w/ group showed marked skin reaction from day 2. Erythema, oedema, and atonia were noted. The severity of the skin irritation increased and by day 7 the majority of animals had hardening and browning of the treated skin. During week 2 fissuring was noted in 5/5 males and 1/5 female animals of the 2.0% w/ group. From day 8, dosing was ceased for various animals in this group (at different stages) due to the condition of the treated skin, including peeling and bleeding.

In the 0.2% w/v group, animals with slight to moderate erythema and atonia were noted from week 1. During week 2, slight to marked hardening (4/10 animals) and fissuring (7/10 animals) were observed. Even after reducing the amount of test substance administered at week 3 (from 2ml/kg to 5ml/animal), the skin reaction increased with slight to marked atonia and fissuring present in all animals; skin hardening, eschar and exfoliation were also prominent. Several animals were not treated or received a smaller dose owing to the condition of the skin. From weeks 5 to 6 conditions of the treated skin improved with the fissuring regressing; slight to moderate erythema, oedema, atonia, desquamation, eschar and exfoliation were observed. During week 9 fissuring was again noted in three of the female animals and by week 10 was apparent in all but one animal. The severity of the reaction increased with most animals having slight to marked hardening, fissuring and eschar. This continued until the end of the study.

The skin reactions in the 0.02% w/v animals were similar to those in the controls. Slight to moderate erythema and oedema were noted in most animals throughout the study; atonia was noted occasionally.

Effect levels

Dose descriptor:
NOAEL
Remarks:
systemic effects
Sex:
male/female
Basis for effect level:
other: systemic effects not observed
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Gonadal tissues were examined for both gross pathology (ovaries only) and histopathology and no treatment-related effects were detected.

Applicant's summary and conclusion

Conclusions:
Dermal administration of the test substance on rabbits elicited marked skin irritation and dermatitis at dose levels of 0.2 and 2.0% w/v. There was no effect on the skin at a dose level of 0.02% w/v. Systemically, there were no effects at dose levels of 0.02% and 0.2% w/v. Minor hyaline droplet accumulation in the kidney was noted at 2% w/v, however, this was considered to be a non-specific response associated with the dermal irritation and not due to direct toxicity on the kidney. A NO(A)ECL for dermal local toxicity was determined to be 0.02% w/v.
Executive summary:

The objective of this study was to assess any topical and/or systemic toxicity of the test item E3333.01 when repeatedly applied to the shaved, non-abraded skin of the New Zealand White rabbit for 13 weeks.

In this 91 day percutaneous toxicity study, the test substance was administered initially, doses of 0 (control), 0.2% and 2.0% w/v to 5 animals per sex, per dose. However, owing to the severity of the skin irritation at the high dose level, animals in the 2.0% w/v group were sacrificed and necropsied during week 3 of the study. An additional group (0.02% w/v) was added to the study as of week 3, and dosing for this group occurred for 12 weeks only (4 males, 6 females). 

There were no biologically significant clinical signs in dosing groups: 0, 0.02%, and 0.2% w/v, to suggest any effect of treatment. Several of the animals in the 2.0% w/v group were noted to not be eating and were thin. All animals in the 2.0% w/v group, except one, lost weight. There was no apparent effect on body weight of 0.02% and 0.2% w/v animals.

All animals in the 2.0% w/ group showed marked skin reaction from day 2. Erythema, oedema, and atonia were noted. The severity of the skin irritation increased and by day 7 the majority of animals had hardening and browning of the treated skin. During week 2 fissuring was noted in 5/5 males and 1/5 female animals of the 2.0% w/ group. From day 8, dosing was ceased for various animals in this group (at different stages) due to the condition of the treated skin, including peeling and bleeding. In the 0.2% w/v group, animals with slight to moderate erythema and atonia were noted from week 1. During week 2, slight to marked hardening (4/10 animals) and fissuring (7/10 animals) were observed. Even after reducing the amount of test substance administered at week 3 (from 2ml/kg to 5ml/animal), the skin reaction increased with slight to marked atonia and fissuring present in all animals; skin hardening, eschar and exfoliation were also prominent. Several animals were not treated or received a smaller dose owing to the condition of the skin. From weeks 5 to 6 conditions of the treated skin improved with the fissuring regressing, slight to moderate erythema, oedema, atonia, desquamation, eschar and exfoliation were observed. During week 9 fissuring was again noted in three of the female animals and by week 10 was apparent in all but one animal. The severity of the reaction increased with most animals having slight to marked hardening, fissuring and eschar. This continued until the end of the study. The skin reactions in the 0.02% w/v animals were similar to those in the controls. Slight to moderate erythema and oedema were noted in most animals throughout the study; atonia was noted occasionally. There was no apparent effect of treatment on any hematology or clinical chemistry parameters. 

In the 0.2% and 2.0% w/v groups there was a dose-related dermatitis. At necropsy this appeared as thickening of the skin with fissuring and in 2.0% group animals was accompanied by sloughing of the superficial layers. 

In the 0.2% w/v group, dermatitis was observed and in the 2.0% w/v group, incomplete re-epithelialization of the tissue underlying the sloughed epidermal layers was observed.  In addition, minor hyaline droplet accumulation in the kidney was observed in one female from the 0.2% group and all animals in the 2.0% w/v group. This was noted as being secondary to dermal irritation and not a direct toxic effect on the kidney. The high dose group (2% w/v) was not considered in the NOAEL determination because this group was sacrificed at week 3 because of skin irritation.

The NO(A)EC for dermal local toxicity was determined to be 0.02% w/v active substance.