Registration Dossier

Administrative data

Description of key information

The repeated dose toxicity of benoxacor has been investigated in oral sub-chronic and chronic studies in rats and dogs and in a sub-acute dermal toxicity study in rabbits. There was no evidence of specific target organ toxicity in any of the studies that would warrant classification or labelling for this end-point.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 March 1990 to 6 March 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hazleton Research Products Inc., Denver, Pennsylvania 17517, USA
- Age at study initiation: 6-7 months
- Weight at study initiation: 6.35-9.80 kg (males); 5.45-7.75 kg (females)
- Fasting period before study: none
- Housing: individually during the working day and in pairs of same sex and group overnight (after removal of food)
- Diet: 400 g/dog/day of SQC Laboratory Diet A, Expanded, pellets (Special Diets Services Ltd., Witham, UK) ad libitum (except overnight prior to blood collection and necropsy)
- Water: filtered mains drinking water ad libitum
- Acclimation period: minimum of 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 16-22°C
- Humidity: 40-80%
- Air changes: minimum of 10/hour
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 1 March 1990 To: 6 March 1991
Route of administration:
oral: capsule
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Calculations of the required amount of GCA154281 to be dosed were based on each dog's weight from the previous week. The correct amount of test substance was weighed and placed into empty gelatin capsules. Capsules were prepared weekly for each individual dog and prior to dosing were stored in a sealed container at ambient temperature.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
52 weeks
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0 (control), 1, 5, 40, 80 mg/kg/day
Basis:
other: nominal
No. of animals per sex per dose:
4
Control animals:
other: yes: empty capsule only
Details on study design:
- Dose selection rationale: based on results from a 28 day oral capsule administration toxicity study in the beagle dog where dose levels of 80, 100, 200 and 300 mg/kg/day were dosed and 80 mg/kg/day was the maximum tolerated dose in this pilot study.
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily before feeding. Additional observations were made throughout the working day as necessary.

DETAILED PHYSICAL EXAMINATION: Yes
- Time schedule: Pre-dose and during weeks 12, 25 and 52

AUDITORY RESPONSE: Yes
- Time schedule: pre-dose and in weeks 13, 26 and 52

BODY WEIGHT: Yes
- Time schedule: weekly throughout the study

FOOD CONSUMPTION: Yes
- Time schedule: daily

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: prior to treatment and during weeks 13, 26 and 52
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to treatment and during weeks 13/14/15, 26 and 52. Samples were collected prior to dosing.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters examined: haemoglobin, mean cell volume, erythrocyte count and indices (mean cell haemoglobin, mean cell haemoglobin concentration, packed cell volume), total and differential white blood cell count, platelet count, Heinz bodies, prothrombin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to treatment and during weeks 13, 26 and 52. Samples were collected prior to dosing.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters examined: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transferase, creatine phosphokinase, sodium, potassium, chloride, inorganic phosphorus, total protein, total lipids, albumin, globulin, A/G ratio, calcium, total bilirubin, urea, creatinine, glucose, total cholesterol.

URINALYSIS: Yes
- Time schedule for collection of urine: prior to treatment and during weeks 13, 26 and 52 by direct catheterisation of the bladder. Samples were collected prior to dosing.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all
- Parameters examined: specific gravity, protein, ketones, blood, reducing substances, pH, glucose, total bilirubin, urobilinogen, colour, turbidity, microscopy of sediment

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all dogs given a complete necropsy)
ORGAN WEIGHTS: Yes
- Organs weighed: adrenals, heart, liver, pituitary, spleen, brain, kidneys, ovaries, testes with epididymes, thyroids.
HISTOPATHOLOGY: Yes (all animals)
- Tissues/organs: all gross lesions, brain (with brainstem), gallbladder, adrenals, aorta, rib and bone marrow, caecum, colon, duodenum, epididymes, eyes (with optic nerve), oesophagus, heart, ileum, jejunum, kidneys, liver, lungs (with mainstem bronchi), lymph nodes (mandibular, popliteal and mesenteric), ovaries, pancreas, parathyroid glands, prostate, sciatic nerve, pituitary, rectum, salivary glands (submaxillary), spinal cord (lumbar, cervical, thoracic), spleen, sternum (with bone marrow), skeletal muscle (Quadriceps), stomach, testes, epididymes, thymus, thyroids (with parathyroids), tongue, trachea, urinary bladder, skin and mammary gland, uterus and vagina.
Statistics:
All variables apart from organ weights were analysed using 2-way analysis of variance (ANOVA). Pairwise comparisons for each sex separately were made using Dunnett's test apart from pre-dose variables for which pair-wise comparisons were made using a protected t-test. Levene's test for equality of variances across groups, between sexes and for any interaction were also performed. When these tests showed evidence of group effects or a sex-group interaction, the data were re-analysed using non-parametric methods for each sex separately. These were Kruskal-Wallis (ANOVA), Wilcoxin rank sum test (pair-wise comparisons) and Terpstra-Jonckheere (dose response) test, where appropriate. If Levene's test showed evidence of differing variances between the sexes only then a one-way ANOVA, regression (if applicable) and Dunnett's (or t-tests) were performed for each sex separately. Organ weights were analysed using analysis of covariance (ANCOVA) using the necropsy bodyweight as a covariate.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: No treatment-related effects

BODY WEIGHT AND WEIGHT GAIN: No toxicologically significant effects. Body weight gain at 40 and 80 mg/kg/day males was lower than male controls at times during the study. The differences were only statistically significant at 80 mg/kg/day for the period weeks 13 - 26.

FOOD CONSUMPTION: No treatment-related effects.

OPHTHALMOSCOPIC EXAMINATION: No treatment-related effects.

AUDITORY RESPONSE: No treatment-related effects.

HAEMATOLOGY: Haemoglobin, red cell count and packed cell volume were lower than controls for 80 mg/kg/day males. Haemoglobin and packed cell volume were also reduced for 40 mg/kg/day males at week 52 and mean cell volume and mean cell haemoglobin for 80 mg/kg/day males were reduced at week 52.

CLINICAL CHEMISTRY: Creatine phosphokinase activities were lower for male treated groups during weeks 13 and 26 and were reduced for female treated groups at 40 and 80 mg/kg/day in week 52. Creatinine levels were lower than controls for males at 40 and 80 mg/kg/day during weeks 13, 26 and 52 and for females at 40 and 80 mg/kg/day in week 52. Bilirubin was increased compared to controls for 80 mg/kg/day males and females in week 13 and for 40 mg/kg/day females and 80 mg/kg/day males and females in weeks 26 and 52.

URINALYSIS: No treatment-related effects

ORGAN WEIGHTS: Absolute and adjusted kidney and liver weights at 40 and 80 mg/kg/day (males and females) were generally higher than controls.

GROSS PATHOLOGY: No treatment-related effects

HISTOPATHOLOGY: 80 mg/kg/day males showed a slight increase compared to controls in the deposition of pigment in kidney proximal tubular cells.
Dose descriptor:
NOEL
Effect level:
5 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Reduced body weight gain, minor disturbances of clinical pathology parameters and increases in liver and kidney weights with pigment deposition in the kidney at 40 and/or 80 mg/kg/day
Critical effects observed:
not specified

Table 1: Intergroup comparison of body weight change (kg) – males

weeks

Dose level of CGA154281 (mg/kg/day)

0

1

5

40

80

-1 to 13

1.79

2.21

1.62

1.61

1.26

13 to 26

0.69

0.55

0.80

0.36

0.06*DR

26 to 39

0.28

0.16

0.23

-0.01

0.38

39 to 52

-0.04

0.04

-0.05

-0.23

-0.06

* Statistically significant difference from control group mean, p<0.05

DR + significant using the dose-response test

 

Table 2: Intergroup comparison of selected haematology parameters

 

Parameter

Dose level of CGA154281 (mg/kg/day)

males

females

0

1

5

40

80

0

1

5

40

80

haemoglobin

wk 13

16.5

16.3

15.0

15.4

14.9*DR

16.3

15.1

16.2

15.4

15.1

wk 26

16.8

17.2

16.0

15.8

14.6*DR

17.1

14.7

16.6

15.3

15.4

wk 52

17.6

17.7

16.7

15.6*

15.1**

17.5

16.4

17.4

16.4

15.8

red cell count

wk 13

7.05

7.12

6.34

6.68

6.41*DR

6.93

6.55

6.93

6.59

6.43

wk 26

7.10

7.32

6.63

6.77

6.35*DR

7.14

6.16

7.01

6.45

6.51

wk 52

7.25

7.43

6.79

6.58

6.52**DR

7.21

6.90

7.14

6.92

6.64

packed cell volume

wk 13

49.0

48.7

44.4

45.4

44.0*DR

48.2

44.8

47.8

45.7

44.7

wk 26

49.2

49.9

46.9

45.7

42.8**DR

49.6

42.3

48.7

44.8

45.0

wk 52

49.9

50.3

47.2

44.4*

43.1**

49.3

46.6

49.3

46.9

45.1

mean cell volume

wk 13

69.5

68.4

70.2

68.1

68.6

69.5

68.5

69.0

69.4

69.5

wk 26

69.2

68.2

70.7

67.5

67.3

69.5

68.7

69.7

69.5

69.1

wk 52

68.9

67.7

69.8

67.5

66.2*DR

68.4

67.6

69.0

67.9

68.0

mean cell haemoglobin

wk 13

23.5

22.9

23.7

23.1

23.2

23.4

23.1

23.3

23.4

23.5

wk 26

23.6

23.5

24.1

23.4

23.0

24.0

23.9

23.8

23.8

23.7

wk 52

24.3

23.9

24.6

23.7

23.2*DR

24.3

23.8

24.4

23.7

23.8

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

DR significant using the dose-response test

 

Table 3: Intergroup comparison of selected clinical chemistry (blood) parameters

 

Parameter

Dose level of CGA154281 (mg/kg/day)

males

females

0

1

5

40

80

0

1

5

40

80

creatine phospho-kinase

wk 13

199

136

134

135

108*DR

153

127

194

132

169

wk 26

227

150***

146***

156***

140***

182

149

165

152

146

wk 52

137

136

119

173

147

199

163

157

123

126*DR

creatinine

wk 13

81

78

79

71

71*DR

77

76

76

72

72

wk 26

96

88

84

75**

80*

81

87

82

75

80

wk 52

86

83

79

69

77*DR

84

100

79

71

70*DR

bilirubin

wk 13

2.0

1.8

1.9

1.9

2.6*DR

2.4

1.8

2.1

2.3

2.9*DR

wk 26

2.9

2.6

2.6

2.8

3.4

2.8

2.4

2.6

3.2

3.7*

wk 52

2.4

2.5

2.7

2.7

3.2*

2.8

2.6

2.7

3.6

3.5**DR

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

*** Statistically significant difference from control group mean, p<0.001

DR significant using the dose-response test

 

 

Table 4: Intergroup comparison of selected organ weights adjusted to final bodyweight

 

Organ

Dose level of CGA154281 (mg/kg/day)

males

females

0

1

5

40

80

0

1

5

40

80

Liver

296.9

327.5

304.0

421.3**

392.0*

249.8

250.3

280.8

354.2*

325.3

Kidney

50.408

52.299

56.188

61.400**

60.749

37.457

43.124

38.352

44.443

43.442

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

 

Table 5: Intergroup comparison of selected histopathological findings in kidney – males

 

Finding

Dose level of CGA154281 (mg/kg/day)

0

1

5

40

80

pigment

1

2

1

2

4

 

Conclusions:
Oral administration of benoxacor tech to the dog for 52 weeks at dose levels up to 80 mg/kg/day was well tolerated.
The NOEL to male and female dogs was 5 mg/kg/day.
Executive summary:

Benoxacor tech was dosed to groups of 4 male and 4 female beagle dogs (oral administration by capsule) for 52 weeks at dose levels of 0 (control), 1, 5, 40 and 80 mg/kg/day.

Oral administration of benoxacor tech to the dog for 52 weeks at dose levels up to 80 mg/kg/day was well tolerated. Changes associated with treatment included a reduced body weight gain, minor disturbances of blood clinical pathology parameters and increases in liver and kidney weights with pigment deposition in the kidney at dose levels of 40 and/or 80 mg/kg/day.

The NOEL to male and female dogs was 5 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
5 mg/kg bw/day
Study duration:
chronic
Species:
dog
Quality of whole database:
A set of studies on the potential long-term toxicity of the substance following repeated-dose oral exposure is available. These studies provide a good understanding of this endpoint and give evidence that benoxacor does not exhibit target organ specific toxicity.

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
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 January 1986 to 14 February 1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
occlusive dressing used
Qualifier:
according to
Guideline:
EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source of animals: H.A.R.E Inc., Hewitt, NJ, USA
- Age at study initiation: 106-113 days
- Weight at study initiation: males 2.86-3.65 kg; females 2.90-3.43 kg
- Fasting period before study: No
- Housing: Individually
- Diet: Certified Purina Rabbit Chow® #5322 ad libitum
- Water: Tap water ad libitum
- Acclimation period: Approximately 36 days

ENVIRONMENTAL CONDITIONS
- Temperature: 65±5°F
- Humidity: 50±20%
- Air changes (per hr): Not reported
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 22 January 1986 To: 14 February 1986
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: Approximately 120-240 cm2
- % coverage: Approximately 5-10% of total body surface
- Type of wrap if used: Gauze dressing secured with non-irritating adhesive tape.
- Time intervals for shavings or clippings: Prior to application and as necessary during the study

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Tap water followed by drying with paper towel
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0 (purified water only), 1, 500, 1010 mg/kg/day
- For solids, paste formed: yes (purified water)

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (Elizabethan collars)
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
22 consecutive days
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
0, 1, 500 and 1010 mg/kg/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during pre-dose period, prior to and regularly after dosing on day 1; prior to dosing, within 30 mins of dosing and 30 mins after end of dosing period, prior to termination

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Pre-dose, prior to dosing and approximately 30 mins after each application period

BODY WEIGHT: Yes
- Time schedule for examinations: Days -8, pre-dose on day 1, weekly thereafter and prior to necropsy

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Pre-dose Days -8 to 1, weekly thereafter

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Day -7 and Day 22
- Dose groups that were examined: All

PHYSICAL/AUDITORY EXAMINATIONS: Yes
- Time schedule for examinations: Day -14 and Day 20

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Days -7, -6 or -3, test days 21, 22, 23 or 24
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (16-18 hours)
- How many animals: All
- Parameters examined: Haemoglobin, haematocrit, erythrocytes, leukocytes, differential leukocytes, platelets, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Days -7, -6 or -3, test days 21, 22, 23 or 24
- Animals fasted: Yes (16-18 hours)
- How many animals: All
- Parameters examined: Urea nitrogen, creatinine, SGOT, SGPT, alkaline phosphatase, glucose, total protein, albumin, globulin, A/G ratio, total bilirubin, total cholesterol, inorganic phosphate, sodium, calcium, potassium, chloride

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all animals) on day 22 or 23

ORGAN WEIGHTS: Yes (all animals)
- Organs weighed: adrenal glands, brain, heart, kidney, liver, ovary, pituitary, testis

HISTOPATHOLOGY: Yes (all animals)
- Organs/tissues examined: Liver, kidney, organs showing gross lesions or changes in size, skin (treated and untreated)
Statistics:
Body weight, food consumption, haematology, clinical chemistry and organ weight data were analysed for each sex separately. Tests for outliers and tests for homogeneity of variance were done. If the model assumptions were met, Dunnett's test was used. Otherwise appropriate ad-hoc analyses including data transforms, nonparametric tests and tests without assuming the homogeneity of group variance were performed. Nonparametric tests also used when parameters known not to be normally distributed.

Microscopic findings analysed separately for each sex by Fisher's Exact test and for both sexes by computing the convolved probabilities.
Clinical signs:
no effects observed
Dermal irritation:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: No mortalities. Clinical signs including inappetence, diarrhoea, soft faeces, and few or no faeces were observed in all groups including control groups. Since the incidence was low and no time or dose-relationships were established, the findings were considered not to be treatment-related.

BODY WEIGHT: Body weight development was comparable between all groups. The slight decrease in mean body weight of top dose males was due to one rabbit only (body weight loss of 260g, 8.1%).

CLINICAL CHEMISTRY: There was slightly decreased blood urea nitrogen in top dose males. In the absence of any meaningful correlation to other clinical chemistry parameters, this was considered to be of no toxicological relevance.

ORGAN WEIGHTS: There was a statistically significant decrease of the mean relative pituitary weights in low dose females, which in absence of any microscopic findings and dose-relationship was considered incidental.
Dose descriptor:
NOEL
Effect level:
1 010 other: mg/kg
Sex:
male/female
Basis for effect level:
other: No toxicologically significant treatment-related findings at highest dose tested
Critical effects observed:
not specified
Conclusions:
Dermal administration of benoxacor at dose levels up to 1010 mg/kg revealed no significant treatment-related findings. Therefore, the NOEL for dermal administration in this study was 1010 mg/kg.
Executive summary:
Benoxacor was administered topically to intact skin under occlusive dressings, for 6 hours per day, to groups of rabbits at doses of 1, 500 or 1010 mg/kg/day for a minimum of 22 consecutive days. Standard parameters were investigated (clinical signs, dermal observations, body weights, food consumption, haematology, clinical chemistry, ophthalmoscopy, organ weights and gross pathological examinations). No treatment-related dermal effect, gross pathology changes or microscopic abnormalities were found. Benoxacor was found to be essentially non-toxic when administered topically at dose levels of up to 1010 mg/kg/day. The no observed effect level (NOEL) was considered to be 1010 mg/kg/day.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 010 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
One reliable, guideline- and GLP-conform study available.

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 January 1986 to 14 February 1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
occlusive dressing used
Qualifier:
according to
Guideline:
EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source of animals: H.A.R.E Inc., Hewitt, NJ, USA
- Age at study initiation: 106-113 days
- Weight at study initiation: males 2.86-3.65 kg; females 2.90-3.43 kg
- Fasting period before study: No
- Housing: Individually
- Diet: Certified Purina Rabbit Chow® #5322 ad libitum
- Water: Tap water ad libitum
- Acclimation period: Approximately 36 days

ENVIRONMENTAL CONDITIONS
- Temperature: 65±5°F
- Humidity: 50±20%
- Air changes (per hr): Not reported
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 22 January 1986 To: 14 February 1986
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: Approximately 120-240 cm2
- % coverage: Approximately 5-10% of total body surface
- Type of wrap if used: Gauze dressing secured with non-irritating adhesive tape.
- Time intervals for shavings or clippings: Prior to application and as necessary during the study

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Tap water followed by drying with paper towel
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0 (purified water only), 1, 500, 1010 mg/kg/day
- For solids, paste formed: yes (purified water)

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (Elizabethan collars)
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
22 consecutive days
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
0, 1, 500 and 1010 mg/kg/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during pre-dose period, prior to and regularly after dosing on day 1; prior to dosing, within 30 mins of dosing and 30 mins after end of dosing period, prior to termination

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Pre-dose, prior to dosing and approximately 30 mins after each application period

BODY WEIGHT: Yes
- Time schedule for examinations: Days -8, pre-dose on day 1, weekly thereafter and prior to necropsy

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Pre-dose Days -8 to 1, weekly thereafter

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Day -7 and Day 22
- Dose groups that were examined: All

PHYSICAL/AUDITORY EXAMINATIONS: Yes
- Time schedule for examinations: Day -14 and Day 20

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Days -7, -6 or -3, test days 21, 22, 23 or 24
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (16-18 hours)
- How many animals: All
- Parameters examined: Haemoglobin, haematocrit, erythrocytes, leukocytes, differential leukocytes, platelets, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Days -7, -6 or -3, test days 21, 22, 23 or 24
- Animals fasted: Yes (16-18 hours)
- How many animals: All
- Parameters examined: Urea nitrogen, creatinine, SGOT, SGPT, alkaline phosphatase, glucose, total protein, albumin, globulin, A/G ratio, total bilirubin, total cholesterol, inorganic phosphate, sodium, calcium, potassium, chloride

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all animals) on day 22 or 23

ORGAN WEIGHTS: Yes (all animals)
- Organs weighed: adrenal glands, brain, heart, kidney, liver, ovary, pituitary, testis

HISTOPATHOLOGY: Yes (all animals)
- Organs/tissues examined: Liver, kidney, organs showing gross lesions or changes in size, skin (treated and untreated)
Statistics:
Body weight, food consumption, haematology, clinical chemistry and organ weight data were analysed for each sex separately. Tests for outliers and tests for homogeneity of variance were done. If the model assumptions were met, Dunnett's test was used. Otherwise appropriate ad-hoc analyses including data transforms, nonparametric tests and tests without assuming the homogeneity of group variance were performed. Nonparametric tests also used when parameters known not to be normally distributed.

Microscopic findings analysed separately for each sex by Fisher's Exact test and for both sexes by computing the convolved probabilities.
Clinical signs:
no effects observed
Dermal irritation:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: No mortalities. Clinical signs including inappetence, diarrhoea, soft faeces, and few or no faeces were observed in all groups including control groups. Since the incidence was low and no time or dose-relationships were established, the findings were considered not to be treatment-related.

BODY WEIGHT: Body weight development was comparable between all groups. The slight decrease in mean body weight of top dose males was due to one rabbit only (body weight loss of 260g, 8.1%).

CLINICAL CHEMISTRY: There was slightly decreased blood urea nitrogen in top dose males. In the absence of any meaningful correlation to other clinical chemistry parameters, this was considered to be of no toxicological relevance.

ORGAN WEIGHTS: There was a statistically significant decrease of the mean relative pituitary weights in low dose females, which in absence of any microscopic findings and dose-relationship was considered incidental.
Dose descriptor:
NOEL
Effect level:
1 010 other: mg/kg
Sex:
male/female
Basis for effect level:
other: No toxicologically significant treatment-related findings at highest dose tested
Critical effects observed:
not specified
Conclusions:
Dermal administration of benoxacor at dose levels up to 1010 mg/kg revealed no significant treatment-related findings. Therefore, the NOEL for dermal administration in this study was 1010 mg/kg.
Executive summary:
Benoxacor was administered topically to intact skin under occlusive dressings, for 6 hours per day, to groups of rabbits at doses of 1, 500 or 1010 mg/kg/day for a minimum of 22 consecutive days. Standard parameters were investigated (clinical signs, dermal observations, body weights, food consumption, haematology, clinical chemistry, ophthalmoscopy, organ weights and gross pathological examinations). No treatment-related dermal effect, gross pathology changes or microscopic abnormalities were found. Benoxacor was found to be essentially non-toxic when administered topically at dose levels of up to 1010 mg/kg/day. The no observed effect level (NOEL) was considered to be 1010 mg/kg/day.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rabbit
Quality of whole database:
One reliable, guideline- and GLP-conform study available.

Additional information

Key studies are considered to be the oral sub-chronic and chronic studies in the rat (Osheroff, 1986a; Ryle, 1991) and dog (Osheroff, 1986b; Wood, 1992).

Rats were fed diet containing 0 (control), 10, 100, 300, 1000 or 6000 ppm benoxacor (0, 0.65, 6.4, 19.0, 63.8 and 417.6 mg/kg/day in males; 0, 0.79, 8.1, 24.1, 79.8 and 492.9 mg/kg/day in females) for 13 weeks. Reductions in body weight gain and food consumption, changes in clinical chemistry and haematology parameters and histopathological findings in the liver were seen at 1000 and 6000 ppm. In the chronic study in rats diets containing 0 (control), 10, 50, 500 and 1000 ppm benoxacor were administered for 52 weeks. Treatment-related findings were confined to reduced body weight gain, food consumption and food utilisation in males and females at 500 and 1000 ppm. In males only at 1000 ppm liver weight was increased marginally with no associated pathological change.

The relevant NOAELs for the rat were: subchronic toxicity 300 ppm (19 and 24.1 mg/kg/day for males and females, respectively); chronic toxicity 50 ppm (2.4 and 3.3 mg/kg/day for males and females, respectively).

Dogs were administered CGA154281 as neat material in gelatine capsules. Doses of 0, 0.25, 1, 5, 50 or 150 mg/kg/day were administered in a 90 day study (Osheroff, 1986b) and 0, 1, 5, 40 and 80 mg/kg/day in a 1 year study. No significant target organ toxicity was seen in either study. Treatment-related effects were generally confined to minor changes in haematology and clinical chemistry parameters and increased liver weight at doses of 40 mg/kg/day and above. In the 1 year study kidney weight was also increased at 40 and 80 mg/kg/day accompanied by a slight increase in pigment deposition in the kidney proximal tubular cells in males only at 80 mg/kg/day.

The relevant NOAELs for the dog were: subchronic toxicity 50 mg/kg/day; chronic toxicity 5 mg/kg/day.

No significant treatment-related toxicity was seen in rabbits dosed dermally for 6 hours/day for 22 consecutive days at doses up to 1010 mg/kg/day (Schiavo, 1986).


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Reliable, guideline- and GLP-conform sub-chronic and chronic oral studies in dogs and rats are available. The chronic study giving the lowest NOAEL was chosen for endpoint characterisation.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
One reliable, guideline- and GLP-conform study available.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
One reliable, guideline- and GLP-conform study available.

Justification for classification or non-classification

The repeated dose toxicity of benoxacor has been investigated in oral sub-chronic and chronic studies in rats and dogs and in a sub-acute dermal toxicity study in rabbits. The available studies on repeated dose toxicity are considered adequate and reliable for the purposes of risk assessment, classification and labelling.

No evidence of significant specific target organ toxicity was seen in repeat dose oral studies in rats and dogs of up to 1 year duration or in a rabbit repeat dose dermal toxicity study.