long chain alkyl thio carbamide metal complex

Endpoint summary

Description of key information

The subacute oral NOAEL is 1000 mg/kg bw/day.
The subacute dermal NOAEL is 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 considered to be 100 mg/kg bw/day, however after correction for purity the local NOAEL is considered to be 67 mg/kg bw/day. 

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03-Feb-1999 to 14-Apr-1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(R) BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, UK
- Age at study initiation: ~8 weeks
- Weight at study initiation: males 255-333 g, females 181-243 g
- Fasting period before study: no data
- Housing: 5/cage; suspended cages with wire mesh floors
- Diet (e.g. ad libitum): SDS pelleted Rat and Mouse No.1, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 28 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-25.5
- Humidity (%): 26-67
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 03-Feb-1999 To: 14-Apr-1999

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- test material administered as a suspension
- suspensions for each dose level prepared independently
- suspensions prepared weight/volume and homogenised
- suspensions prepared daily for the first 7 days then every 2 days, stored in amber glass bottles at 4 ºC

VEHICLE
- Concentration in vehicle: no data, but presumably 3, 30 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the commencement of the study, the proposed formulation procedure was checked by chemical analysis to confirm that the method was acceptable and that the stability and homogeneity of the formulation was satisfactory under the conditions of the study. Samples of the formulations prepared for use in weeks I and 3 were also analysed to check the accuracy of preparation. Results reported separately.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Peanut oil only

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/sex in 0 and 1000 mg/kg bw/day groups
5/sex in 15 and 150 mg/kg bw/day groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: with reference to existing toxicity data, including a 7-day preliminary study with dose levels of 250, 500 and 1000 mg/kg bw/day
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: random at start of study
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): no data

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations included: signs of reaction to treatment or ill health

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
- detailed physical examination: once weekly
- detailed observations in association with dosing: daily during week 1, twice weekly during weeks 2-4; observations were made pre-dose and four times post-dosing

BODY WEIGHT: Yes
- Time schedule for examinations: study days 1, 8, 15, 22, 28 and recovery days 1, 8 and 14; also at necropsy

FOOD CONSUMPTION: Yes
- food consumption calculated as weekly group mean consumption (g/rat per week) = [(total food given to group - total food left by group) / (number of animal days for the group)] * 7
- quantity of food consumed was recorded weekly for each cage

FOOD EFFICIENCY:
- food conversion (%) = mean food consumption (g) / mean body weight gain (g)
- calculated on a weekly basis

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: main study day 29, recovery animals day 43
- Anaesthetic used for blood collection: Yes, light general anaesthesia
- Animals fasted: Yes
- How many animals: all
- Parameters examined: packed cell volume, haemoglobin, red blood cell count, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration, mean corpuscular volume, differential white blood cell count (neutrophils, lymphocytes, eosinophils, basophils, monocytes, large unstained cells), total white blood cell count, platelet count, prothrombin time, activated partial thromboplastin time, bone marrow smear

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: main study day 29, recovery animals day 43
- Animals fasted: Yes
- How many animals: all
- Parameters examined: glucose, total protein, albumin, albumin/globulin ratio, urea nitrogen, creatinine, alkaline phosphatase, glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase, gamma-glutamyltranspeptidase, bilirubin, sodium, potassium, calcium, inorganic phosphorus, chloride, cholesterol, triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: main study day 29, recovery animals day 43
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: appearance, volume, pH, specific gravity, protein, glucose, ketones, bile pigments, haem pigments, crystals, epithelial cells, leukocytes, erythrocytes, casts, other abnormal components (including spermatozoa and precursors)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to the start of treatment, during week 4 and during week 2 of recovery
- Dose groups that were examined: all
- Battery of functions tested: full functional observational battery and quantitative assessment of locomotor activity
- Other: examinations in the hand and standard arena were also performed on all main study rats during weeks 2 and 3

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

adrenals# w
alimentary tract (oesophagus, stomach*, duodenum*, jejunum*, ileum*, caecum*, colon*, rectum *)
aorta (thoracic)
brain* w
epididymides# w
eyes
femur#
gross abnormalities#
harderian glands*
heart* w
kidneys# w
lachrymal gland
liver# w
lungs (including bronchi)*
lymph nodes (mandibular, mesenteric)*
mammary area (caudal)
marrow smear
optic nerves
ovaries* w
pancreas
pituitary
prostate* w
salivary gland
sciatic nerve
seminal vesicles* w
skeletal muscle (thigh)
skin (overlying mammary area)
spinal cord*
spleen# w
sternum
testes# w
thymus*
thyroids (with parathyroids)*
tongue
trachea*
urinary bladder*
uterus (with cervix)*
vagina*

w - indicates organ weighed
* - indicates tissues examined for all main study animals
# - indicates tissues examined for main study animals in control and top dose groups

Other examinations:

no data

Statistics:

Fisher's Exact Test and Mantel's test for data where the relative frequency of the mode exceeded 75%
Bartlett's test for heterogeneity of variance; if significant, logarithmic transformation tried
One-way analysis of variance if non-significant Bartlett's; Kruskal-Wallis analysis if significant Bartlett's
Student's t-test and Williams test or non-parametric equivalents where appropriate

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clear or brown coloured saliva observed immediately after dosing, this was considered to be associated with oral gavage administration.
Incidental hair loss from various regions of the body was also observed in a proportion of the rats from all groups during the study.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight gain for group 4 (1000 mg/kg/day) males was statistically significantly lower than the vehicle control. The body weight gain was also lower for both sexes of group 3 (150 mg/kg/day) and male group 2 (15 mg/kg/day). As the differences in body weight, particularly for males, were more marked in the lower doses than the high dose, and there was some evidence of similar differences in growth rates prior to the start of treatment, the bodyweight differences recorded during the dosing period were considered to be unrelated to test item treatment.
Over the 2 week recovery period, the bodyweight gain for group 4 males was slightly lower than the vehicle control, but not statistically different. Group 4 females were similar to the control.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The differences between the control and treated groups were considered not to be treatment-related.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At week 5, prothrombin for group 3 and group 4 males were statistically higher than the vehicle control. These differences were minimal, did not show a relationship to dose and were only present in one sex, therefore these were considered unrelated to treatment.
Week 5 neutrophil and platelet counts for group 4 females were statistically significantly higher than vehicle control. All values were within the range of the background data for rats of the age and strain used, therefore, these findings were considered unrelated to treatment.
At week 7 all parameters were within accepted limits.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At week 5, urea nitrogen for group 4 females was statistically significantly higher than the vehicle control. This difference was minimal, present in one sex and did not show a relationship to dose. Therefore, this finding was considered unrelated to treatment.
At week 7, creatine and calcium level in group 4 males, and glucose and triglyceride levels in group 4 females were statistically significantly lower than the vehicle control. The albumin/globulin ration in group 4 males were statistically significantly higher than the vehicle control. However, these differences were minimal, present in one sex and were only evident 2 weeks after cessation of treatment. Therefore, these findings were considered incidental and unrelated to treatment.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

At week 5, the pH for group 4 males was statistically significantly lower than the vehicle control. A mild dose response relationship was evident, however the change was small, confined to one sex and all values were within the range of the background data for rats of the age and strain used. For these reasons, the observed differences were considered to be unrelated to treatment.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

At week 5, liver weights for group 4 females were statistically significantly higher than the vehicle control, however this difference was present only in one sex and all individual values were within the normal background range for rats of the age and strain. Therefore this was considered to be unrelated to treatment.
Relative spleen weight to bodyweight values for group 4 females were statistically significantly higher than the vehicle control. However, in the absence of statistically significant result for absolute organ weights, and there was some overlap between the two groups, this observation is considered unrelated to treatment.
At week 7, kidney weights (adjusted for, and relative to, bodyweight) for group 4 males and relative heart weight to bodyweight values for group 4 females were statistically significantly higher than those for the vehicle control. Since these differences were only evident 2 weeks are cessation of treatment, they were considered incidental and unrelated to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Forestomach - brown staining on the epithelial aspect, 3/5 males and 4/5 females in the top-dose group and 1/5 females in the mid-dose group
Large intestine - dark contents, all animals in top-dose group
These findings are considered to be pigmentation associated with the coloured nature of the test material
Recovery animals showed no changes attributable to treatment
Incidence and distribution of all other findings were considered to fall within the background range of macroscopic changes.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Mesenteric lymph nodes - eosinophilic/vacuolated macrophages were seen in top-dose males (5/5) and females (5/5), p<0.01, at the end of the 4-week treatment period and after a 2-week recovery period. This change showed partial reversibility in rats allowed a 2-week recovery period. This finding is not considered an adverse response but indicates that some material was absorbed through the gastro-intestinal tract and taken up in the mesenteric lymph nodes.
All other microscopic findings seen in rats at the end of the treatment period were considered to be incidental and of no toxicological importance
- no microscopic findings were detected which might be associated with the statistically higher relative liver weights for top-dose females; therefore this finding was considered to be incidental.
- no microscopic findings were seen in the gastrointestinal tract of any animals in this study.

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
- there were no clinical signs associated with a toxic response to the test material.
- clear or brown coloured saliva was observed immediately after dosing in some animals from all treated groups. This was considered to be associated with oral gavage administration of a bolus dose of the test material formulated in peanut oil.
- incidental hair loss from various regions of the body (with or without scabbing) was observed in some animals from all groups

BODY WEIGHT AND WEIGHT GAIN
- during the 4-week dosing period, body weight gain in top-dose males, mid-dose males and females and low-dose males was statistically significantly reduced. The differences, particularly for the males, were more marked at the lower doses than at the high dose, and were considered to reflect lower food consumption noted for these groups. Furthermore, there was some evidence of similar differences in growth rates prior to the start of treatment. As such, the body weight differences recorded during the dosing period were considered to be unrelated to treatment.
- during the 2-week recovery period, body weight gain for top-dose males was slightly reduced but the difference was not statistically significant.

FOOD CONSUMPTION
- Cumulative food consumption in the top-dose groups were similar to control during the dosing and recovery periods. Reduced food consumption was seen in low-dose males and mid-dose females during the 4-week dosing period, and differences between control and treated groups in cumulative food consumption were considered not to be treatment-related

FOOD EFFICIENCY
- food conversion ratios were variable with no evidence of a treatment-related effect

HAEMATOLOGY
- week 5, prothrombin time, mid- and high-dose males, statistically significantly increased. These differences were minimal, did not show a relationship to dose, were present only in one sex and, therefore, were considered unrelated to treatment.
- week 5, neutrophil and platelet counts, top-dose females, statistically significantly increased. A slight numerical dose relationship was evident for the neutrophils, however all values were within the range of background data for rats of the age and strain used. These findings are therefore considered to be unrelated to treatment.
- week 7, all parameters were within accepted limits and there were no statistically significant differences between top-dose groups and vehicle control.
- remaining parameters measured, but not mentioned above, were within accepted limits and showed no evidence of a response to treatment.

CLINICAL CHEMISTRY
- week 5 in the main study, urea nitrogen, top-dose females, statistically significantly increased. However, this difference was minimal, present only in one sex, did not show a relationship to dose and was considered unrelated to treatment. Furthermore, the same group mean (17 mg/dl) value was recorded for both top-dose and vehicle control groups at week 7.
- week 7 in the main study, creatinine and calcium level, top-dose males, statistically significantly reduced; glucose and triglyceride levels, top-dose females, statistically significantly reduced; albumin/globulin ratio, top-dose males, statistically significantly increased. However, these differences were minimal and were present only in one sex. In addition these differences were only evident 2 weeks after the cessation of treatment. Therefore, they were considered incidental and unrelated to treatment.
- remaining parameters measured, but not mentioned above, were within accepted limits and showed no evidence of a response to treatment.

URINALYSIS
- week 5 in the main study, pH in the top-dose males was statistically significantly reduced. A mild dose relationship was evident, however the change was small, confined to one sex and all values were within the range of background data for rats of the age and strain used. For these reasons the observed differences were considered to be unrelated to treatment.
- remaining parameters measured, but not mentioned above, were within accepted limits and showed no evidence of a response to treatment.

NEUROBEHAVIOUR
- no changes considered to be indicative of neurotoxicity

ORGAN WEIGHTS
- week 5 in the main study, relative liver weight, top-dose females, statistically significantly increased. However, this difference was present only in one sex and all individual values were within the normal background range for rats of the age and strain used. This finding is therefore considered to be unrelated to treatment
- week 5 in the main study, relative spleen weight, top-dose females, were also statistically significantly increased. There was some overlap of the individual data between this test group and the controls, and in the absence of a statistically significant result for absolute organ weights the observed differences were considered to be unrelated to treatment
- week 7 in the main study, relative kidney weight, top-dose males, statistically significantly increased; relative heart weight, top-dose females, statistically significantly increased. Since these differences were only evident 2 weeks after the cessation of treatment, they were considered incidental and unrelated to treatment
- remaining organs weighed, but not mentioned above, were within accepted limits and showed no evidence of a response to treatment.

GROSS PATHOLOGY
- forestomach, brown staining on the epithelial aspect, 3/5 males and 4/5 females in the top-dose group and 1/5 females in the mid-dose group
- large intestine, dark contents, all animals in top-dose group
- these findings are considered to be pigmentation associated with the coloured nature of the test material
- recovery animals, no changes attributable to treatment
- incidence and distribution of all other findings were considered to fall within the background range of macroscopic changes.

HISTOPATHOLOGY: NON-NEOPLASTIC
- mesenteric lymph nodes, eosinophilic/vacuolated macrophages were seen in top-dose males (5/5) and females (5/5), p<0.01, at the end of the 4-week treatment period and after a 2-week recovery period. This change showed partial reversibility in rats allowed a 2-week recovery period. This finding is not considered an adverse response but indicates that some material was absorbed through the gastro-intestinal tract and taken up in the mesenteric lymph nodes.
- all other microscopic findings seen in rats at the end of the treatment period were considered to be incidental and of no toxicological importance
- no microscopic findings were detected which might be associated with the statistically higher relative liver weights for top-dose females; therefore this finding was considered to be incidental.
- no microscopic findings were seen in the gastrointestinal tract of any animals in this study.

HISTOPATHOLOGY: NEOPLASTIC:
- No data

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Conclusions:

In a GLP study conducted according to OECD guideline 407, no treatment-related adverse effects were seen in rats administered the test material by gavage at up to 1000 mg/kg bw/day (the highest tested dose) for 28-days, and this dose is considered the NOAEL for this study.

Executive summary:

In a GLP study conducted according to OECD guideline 407, groups of 5 male and 5 female Crl:CD(R) BR rats were administered EC:434 -650 -5 at 0, 15, 150 or 1000 mg/kg bw/day by oral gavage (in peanut oil) for 28 days. Additional groups of 5 rats/sex, administered 0 or 1000 mg/kg bw/day, were retained for a further 2-week recovery period.

There were no deaths reported. Clear or brown coloured saliva was seen immediately after dosing in some animals from all treated groups; this was considered to be associated with the method of dosing. Relative liver weight was statistically significantly increased in females administered 1000 mg/kg bw/day. However, this difference was present only in one sex, all individual values were within the normal background range for rats of the age and strain used, and no associated microscopic findings were detected; it is therefore considered to be unrelated to treatment.

At necropsy, brown staining was observed on the epithelial aspect of the forestomach in 3/5 males and 4/5 females treated with 1000 mg/kg bw/day and 1/5 females treated with 150 mg/kg bw/day, and dark contents were seen in the large intestine of all rats treated with 1000 mg/kg bw/day. These findings were considered to be pigmentation associated with the coloured nature of the test material. Histopathological examination revealed eosinophilic/vacuolated macrophages in the mesenteric lymph nodes of all main study rats treated with 1000 mg/kg bw/day and, to a lesser extent, in recovery rats treated similarly indicating partial reversibility. This finding is not considered an adverse response but indicates that some test material was absorbed through the gastrointestinal tract and taken up in the mesenteric lymph nodes.

In conclusion, the 28-day oral no-observed-adverse-effect level (NOAEL) for EC:434 -650 -5 in rats was 1000 mg/kg bw/day (the highest tested dose).

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Conclusions:

In a GLP study conducted according to OECD guideline 407, no treatment-related adverse effects were seen in rats administered NB 6786-35 by gavage at up to 1000 mg/kg bw/day (the highest tested dose) for 28-days, and this dose is considered the NOAEL for this study.

Executive summary:

In a GLP study conducted according to OECD guideline 407, groups of 5 male and 5 female Crl:CD(R) BR rats were administered NB 6786-35 (tris (dicocodithiocarbamato) tris (µ-disulphido) (µ3-thio)-triangulotrimolybdenum (IV) dicocodithiocarbamate)at 0, 15, 150 or 1000 mg/kg bw/day by oral gavage (in peanut oil) for 28 days. Additional groups of 5 rats/sex, administered 0 or 1000 mg/kg bw/day, were retained for a further 2-week recovery period.

There were no deaths reported. Clear or brown coloured saliva was seen immediately after dosing in some animals from all treated groups; this was considered to be associated with the method of dosing. Relative liver weight was statistically significantly increased in females administered 1000 mg/kg bw/day. However, this difference was present only in one sex, all individual values were within the normal background range for rats of the age and strain used, and no associated microscopic findings were detected; it is therefore considered to be unrelated to treatment.

At necropsy, brown staining was observed on the epithelial aspect of the forestomach in 3/5 males and 4/5 females treated with 1000 mg/kg bw/day and 1/5 females treated with 150 mg/kg bw/day, and dark contents were seen in the large intestine of all rats treated with 1000 mg/kg bw/day. These findings were considered to be pigmentation associated with the coloured nature of the test material. Histopathological examination revealed eosinophilic/vacuolated macrophages in the mesenteric lymph nodes of all main study rats treated with 1000 mg/kg bw/day and, to a lesser extent, in recovery rats treated similarly indicating partial reversibility. This finding is not considered an adverse response but indicates that some test material was absorbed through the gastrointestinal tract and taken up in the mesenteric lymph nodes.

In conclusion, the 28-day oral no-observed-adverse-effect level (NOAEL) for NB 6786-35 in rats was 1000 mg/kg bw/day (the highest tested dose). [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.]

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 February 1998 - 1 May 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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"

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

Specific details on test material used for the study:

67% purity

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

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 clippings: 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

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

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

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 satellite 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 satellite animals
- Parameters checked in Table 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: No

Sacrifice and pathology:

GROSS PATHOLOGY:
A full macroscopic postmortem 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.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related clinical signs of toxicity at any dose level tested.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

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).

Mortality:

no mortality observed

Description (incidence):

All main study and satellite animals survived to terminal sacrifice

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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 and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

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 biochemistry findings:

no effects observed

Description (incidence and severity):

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.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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 pathological findings:

no effects observed

Description (incidence and severity):

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.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.

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.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

670 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

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, post-mortem 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, however, after correction for purity of 67% the local NOAEL is 67 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.

Reference 2

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

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

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

670 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

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, however, after correction for 67% purity the local NOAEL is considered to be 67 mg/kg bw/day.

On the grounds of animal welfare and in view of the similarity of this compound, both chemically and structurally, it is considered the results of this study can be read across to support the notification of EC# 457-320-2.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

670 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 February 1998 - 1 May 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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"

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

Specific details on test material used for the study:

67% purity

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

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 clippings: 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

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

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

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 satellite 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 satellite animals
- Parameters checked in Table 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: No

Sacrifice and pathology:

GROSS PATHOLOGY:
A full macroscopic postmortem 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.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related clinical signs of toxicity at any dose level tested.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

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).

Mortality:

no mortality observed

Description (incidence):

All main study and satellite animals survived to terminal sacrifice

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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 and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

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 biochemistry findings:

no effects observed

Description (incidence and severity):

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.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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 pathological findings:

no effects observed

Description (incidence and severity):

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.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.

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.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

670 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

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, post-mortem 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, however, after correction for purity of 67% the local NOAEL is 67 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.

Reference 2

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

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

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

670 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

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, however, after correction for 67% purity the local NOAEL is considered to be 67 mg/kg bw/day.

On the grounds of animal welfare and in view of the similarity of this compound, both chemically and structurally, it is considered the results of this study can be read across to support the notification of EC# 457-320-2.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

67 mg/cm²

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1

Additional information

No treatment-related systemic adverse effects were seen in a GLP 28-day repeated-dose dermal toxicity study of a substance structurally and chemically similar to EC# 457-320-2. The top dose used was 1,000 mg test material/kg bw/day, and the test material comprised 67% active substance. The NOAEL for systemic effects was therefore 670 mg/kg bw/day. (The NOAEL for local dermal irritation effects was 2 mL/kg of 5% solution, or 100 mg/kg bw/day).

No treatment-related systemic adverse effects were seen in a GLP 28-day repeated-dose oral toxicity study of the same substance structurally and chemically similar to EC# 457-320-2. An increase in liver weight was seen in treated animals, but there was no associated histopathology and the study personnel did not consider it to be a toxicologically-significant effect. The NOAEL was therefore 1,000 mg/kg bw/day, the top dose used.

Justification for classification or non-classification

No treatment-related systemic toxicity was seen in the 28-day dermal toxicity study of a substance structurally and chemically similar to EC# 457-320-2, and the NOAEL was concluded to be 670 mg/kg bw/day. No hazard classifications are required based on this study.

The repeat-dose oral study available (28-day toxicity study) identified no consistent identifiable treatment-related toxic effects at dose levels that might necessitate classification for Specific Target Organ Toxicity (STOT-RE). Consequently, in accordance with CLP, no hazard classifications are proposed.