long chain alkyl thio carbamide metal complex

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

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

Adequacy of study:

key study

Study period:

25 February 1998 - 1 May 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study, to GLP, on read-across material

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

yes

Remarks:

Slight protocol deviations were noted in the study report (including a dosing accident and subsequent replacement animals), but it was stated that "it is unlikely that these protocol exceptions adversely affected the results or integrity of the study"

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Deviations:

yes

Remarks:

as above

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): EXP 974019 (MRD-98-158)
- Substance type: Technical product
- Physical state: Black liquid
- Analytical purity: considered pure for the purpose of dosing
- Expiration date of the lot/batch: February 2003
- Stability under test conditions: Diluted in peanut oil, the test material was stable at room temperature for at least eight days
- Storage condition of test material: Room temperature

Test animals

Species:

rat

Strain:

other: Crl:CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: Males approx 8 weeks; females approx. 9 weeks
- Weight at study initiation: Males 248-275 g; females 200-232 g
- Fasting period before study: none
- Housing: Singly in suspended stainless steel and wire mesh cages
- Diet: ad libitum, PMI Certified Rodent Diet Meal 5002, PMI Feeds Inc, Richmond, Indiana
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-22
- Humidity (%): 30-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): approx. 12/12

IN-LIFE DATES: From: 19 March 1998 To: 1 May 1998

Administration / exposure

Type of coverage:

occlusive

Vehicle:

peanut oil

Details on exposure:

Method of administration:
Dermal

TEST SITE
- % coverage: approx. 10% of total body surface
- Type of wrap if used: porous gauze dressing secured with non-irritating tape and wrapped with COBAN
- Time intervals for shavings or clipplings: approx. 24 h prior to initial administration and at least once weekly

REMOVAL OF TEST SUBSTANCE
- Washing (if done): removed by gently wiping the exposure site with peanut oil and a paper towel.
- Time after start of exposure: at least 6 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0, 100, 300 and 1000 mg/kg bw/day
- Constant volume or concentration used: each dose group used a different concentration, and individual dose volumes were adjusted weekly based on the most recent body weights

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test substance was soluble in peanut oil at the concentrations used in the study
- Amount(s) applied (volume or weight with unit): 2.0 ml/kg bw
- Concentration (if solution): no data

USE OF RESTRAINERS FOR PREVENTING INGESTION: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance in peanut oil were received for analysis at nominal concentrations ranging from 1 % to 75%, diluted with n-heptane to give final nominal concentrations between 0.08% and 0.26% and analyzed for uniformity, stability, and concentration verification using reverse phase High Performance Liquid Chromatography (HPLC). The uniformity of the test substance in peanut oil was evaluated for samples mixed on 18 March 1998, the stability was evaluated over an eight-day period for samples mixed on 18 March 1998 and the concentration verification was evaluated for the samples mixed on 19 March 1998 and 3 April 1998. Satisfactory uniformity was observed, stability data indicated that the test substance diluted in peanut oil was stable at room temperature for at least eight days and concentration verification analyses indicated that the test substance concentration was within 10% of the nominal value.

Duration of treatment / exposure:

28 days

Frequency of treatment:

at least 6 h/day, 7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
nominal per unit body weight

No. of animals per sex per dose:

10/sex/dose in the control and high-dosed groups and 5/sex/dose in the low and mid-dosed groups

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: Based on a 1-week range-finding study which showed no signs of overt toxicity at the limit dose level of 1000 mg/kg bw when diluted in peanut oil. The low- and mid-dose levels were selected to provide a logarithmic progression of dosages.
- Rationale for animal assignment (if not random): After determination of suitability based on health, body weight and other abnormalities, a computer generated sorting programme was used
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily from Monday-Friday and once daily on Saturdays, Sundays and holidays
- Cage side observations included: viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during the test period

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Prior to dosing on days 0, 1, 4, 7, 11, 14, 18, 21 and 25; also after sleeve removal on day 0 and prior to blood collection on day 28; additional dermal evaluations for satellite animals on days 32, 35, 39 and 42. All dermal evaluations performed according to the Draize method of scoring.

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to initiation of dosing for group allocation, on the day of initiation of dosing and on days 7, 14, 21 and 27; body weights for satellite animals also recorded on days 35 and 41.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Main study animals - day 28, satellite animals - day 42
- Anaesthetic used for blood collection: Yes - methoxyflurane
- Animals fasted: Yes
- How many animals: All main study and all satelite animals
- Parameters checked in Table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Main study animals - day 28, satellite animals - day 42
- Animals fasted: Yes
- How many animals: All main study and all satelite animals
- Parameters checked in Table 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: No

Sacrifice and pathology:

GROSS PATHOLOGY:
A full macroscopic postmorten examination performed on all animals, and required organs were preserved.

HISTOPATHOLOGY:
(see table 3 below)

Other examinations:

ORGAN WEIGHTS:
Liver, kidneys, testes, adrenals and brain were weighed from all main study animals on day 28 and all satellite animals on day 42

Statistics:

Bartlett's Test was performed to determine if the dose groups had equal variance (Snedecor and Cochran, 1989). If the variances were equal, the testing was done using parametric methods, otherwise nonparametric techniques were used.

Parametric procedures - a standard one way ANOVA using the F distribution to assess significance was used (Snedecor and Cochran, 1989). If significant differences among the means were indicated, Dunnett's Test was used to determine which treatment groups differed significantly from control (Dunnett, 1964). In addition to the ANOVA, a standard regression analysis for linear response in the dose groups was performed. This also tested for linear lack of fit in the model.

Non parametric procedures - the test of equality of means was performed using the Kruskal-Wallis Test (Hollander and Wolfe, 1973). If significant differences among the means were indicated, Dunn's Summed Rank Test was used to determine which treatment groups differed significantly from the control (Hollander and Wolfe, 1973). In addition to the Kruskal-Wallis Test, Jonckheere's Test for monotonic trend in the dose response was performed (Hollander and Wolfe, 1973).

Bartlett's Test was conducted at the 1% level of significance and all others at the 5% and 1% level of significance.

The satellite group's recovery parameters were compared using the statistical t-test (Dixon and Massey, 1969).

The statistical tests were considered appropriate.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY: All main study and satellite animals survived to terminal sacrifice and showed no treatment-related clinical signs of toxicity at any dose level tested. Incidental findings were limited to occasional scabs among both sexes. Slight transient dermal irritation was the only significant observation and was limited to erythema, desquamation and/or oedema, observed primarily in the top-dosed males and in all groups of females during week 1 and starting on day 4. Erythema and/or oedema was also observed in two control females and desquamation in 3 control females. Irritation was scored according to Draize, 1959. The dermal effects in the 100 and 300 mg/kg bw/day groups did not show a noticeable dose-response difference from the controls (though, treatment-related dermal histopathology was seen in 300 mg/kg bw/day animals).

BODY WEIGHT AND WEIGHT GAIN: There were no statistically significant differences in mean body weight or mean body weight gain between treated and control females at any interval. All male animals showed increases in body weight over their initial values. Mean body weight gain of the high-dosed males was significantly decreased on day 27 when compared with the controls (p≤0.05) and similar significant decreases were also seen between days 0-7 and 14-21. This decrease was transient with the mean body weight being comparable to the controls following the recovery period and was not considered to be biologically or toxicologically important.

FOOD CONSUMPTION: There were no statistically significant differences in mean food consumption between the treated and control main study or satellite animals of either sex.

HAEMATOLOGY: No statistically significant differences in haematological parameters were seen in any of the main study animals. Following the recovery period, mean white blood cells and mean absolute monocytes were significantly decreased in the males when compared with the controls however these were not considered to be clinically significant or related to the test substance.

CLINICAL CHEMISTRY: No biologically meaningful differences in serum chemistry parameters were seen in any of the main study animals and there were no statistically significant differences following the recovery period.

ORGAN WEIGHTS: No differences in mean absolute, relative organ/body, or relative organ/brain weights were seen which were considered to be treatment-related. A statistically significant increase in the mean absolute adrenal weight, mean adrenal:body weight ratio and mean adrenal:brain weight ratio amongst the top-dosed females when compared to the controls in the main study, was not considered of relevance due to the absence of a clear dose response. Following recovery, there was a statistically significant increase in mean relative kidney:body weight ratio among the top-dosed males when compared to the controls however this difference was small and, in the absence of similar findings in the main study, was considered spurious and unrelated to the test substance.

GROSS PATHOLOGY: No gross pathological changes were observed in any organ or tissue to indicate any type of systemic toxicity. Discoloured livers were frequently observed in all groups including the controls in both the main study and following the recovery period. However, this was most likely caused by the wrapping procedure and not considered to be related to the test substance.

HISTOPATHOLOGY: Microscopic changes were seen in the treated skin area of the mid- and high-dosed animals in the main study. These changes consisted of acanthosis and hyperkeratosis of the epidermis and an associated sebaceous gland hyperplasia. A decrease in the degree and incidence of these effects was seen during the recovery phase, indicating that the changes were reversible. Changes were also noted, to a lesser degree, in the control and low-dosed animals and were considered to have been due to the repeated shaving and wrapping of the torso and/or a mild irritating effect of the test substance. No further microscopic changes were observed in any of the other organs or tissues examined.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: original NCD unit is mg/kg/day.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a GLP study conducted in general agreement with OECD guideline 410, the 28-day dermal NOAEL in the rat was 1000 mg/kg bw/day (the highest tested dose), equivalent to 670 mg/kg bw/day active ingredient.

Executive summary:

In a GLP study conducted in general agreement with OECD guideline 410, the repeated dermal toxicity of a compound which is both chemically and structurally similar to EC# 457-320-2 was determined in Crl:CD(R) BR rats. Groups of 5 male and 5 female animals were treated, under covered contact, to the clipped skin with 0, 100, 300 or 1000 mg/kg bw/day (in peanut oil) for 28 days. The residual test substance was removed by gently wiping with peanut oil and a paper towel after at least 6 hours. Additional groups of 5 animals/sex were similarly treated with 0 or 1000 mg/kg bw/day and allowed to recover for 14 days to test for reversibility, persistence or delayed occurrence of toxic effects. Individual doses were adjusted weekly based on the most recent body weights.

Dermal application of the test substance under the conditions of this study elicited no signs of systemic toxicity. There were no adverse clinical signs, postmortem findings, non-dermal treatment-related histopathological findings, biologically significant changes in body weight, food consumption or absolute/relative organ weights, or clinically significant changes in hematology, clotting potential or serum chemistry which were related to treatment. Treatment-related findings were limited to microscopic changes in the treated area of the skin of animals in the 300 and 1,000 mg/kg bw/day groups. All other changes observed were considered to be either incidental or indirectly related to experimental manipulation (i.e. wrapping). The NOAEL for the test material under the conditions of this study was determined to be 1000 mg/kg bw/day. The test material consisted of 67% active substance in refined base oil; therefore, the NOAEL for the active substance was 670 mg/kg bw/day. The NOAEL for local effects at the site of administration is concluded to be 100 mg/kg bw/day.

In view of the structural and chemical similarities, it is considered that the results of this study can be used for read-across to EC# 457-320-2.