Benzododecinium chloride

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Study period:

From June 18, 1987 to June 21, 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals
- Source: Sprague-Dawley CD rats were obtained from Charles River Breeding Laboratories, Portage, MI, USA.
- Age at study initiation: 7 weeks
- Weight at study initiation: 221.6-250.9 g (males); 147.7-174.4 g (females)
- Housing: Animals were housed one animal/side of divided stainless steel cages (solid sides with wire mesh floors) mounted in a stainless steel Maxi-Rack (Hazleton Systems, Inc., Aberdeen, MD).
- Diet: Ground Purina Certified Rodent Chow # 5002 (Ralstor Purina Co., St. Louis, MO), ad libitum
- Water: Tap water, ad libitum. Water was provided by an automatic watering system with demand control valves mounted on each cage rack
- Acclimation period: 1 week

Environmental conditions:
- Temperature: 22 ± 3°C
- Humidity: 40-70%
- Photoperiod: 12h light/12h dark

In-life dates: From: 18 June 1987 to: 22 September 1987

Route of administration:

oral: feed

Vehicle:

other: Ground Purina Certified Rodent Chow # 5002

Details on oral exposure:

Diet preparation: Test diets were prepared by direct addition of the test substance to ground rodent feed. A concentrated premix was prepared to ensure maximal loss of the ethanol (approximately 12% by weight) from the test substance during the original mixing time of 1h. Test diets were prepared by appropriate dilutions of the concentrated premix or higher diet concentrations.
- Rate of preparation of diet (frequency): Fresh diet was prepared and offered to the animals each week.
- Mixing appropriate amounts with (Type of food): Ground Purina Certified Rodent Chow # 5002
- Storage temperature of food: Diets were stored in polypropylene containers at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Experimental diets were analyzed for stability, homogeneity and concentration of test substance in the diet by using liquid chromatography.
- Homogeneity studies performed on samples from all dietary concentrations indicated that test substance was uniformly distributed in the diet.
- Stability studies were conducted on diets (8000 and 100 ppm) stored at room temperature in closed polypropylene containers and in glass jars. These analyses indicated that the test substance was stable in the diets for at least 21 d under both storage conditions for all dose levels.
- Concentration verification analyses revealed that the test substance concentrations were 90.7 to 111.8% of nominal for all 5 concentrations.

Duration of treatment / exposure:

95 and 96 days for males and females, respectively.

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 100, 500, 1000, 4000 or 8000 ppm (equivalent to 0, 6, 31 and 62 mg/kg bw/day (males) and 0, 8, 38 and 77 mg/kg bw/day (females). [Due to mortality in the 4000 and 8000 ppm treatment groups, the corresponding daily intakes could not be calculated.]
Basis: nominal in diet

No. of animals per sex per dose:

15

Control animals:

yes, plain diet

Details on study design:

- Rationale for animal assignment: Animals were assigned to test groups, based on body weight, by a computer generated, weight stratified randomization procedure. Only rats with body weights within ± 20% of the population mean for each sex were used in the study.

Assignment of animals: The animals were assigned into following groups in the study:
- Group 1(Diet control): Plain diet
- Group 2: 100 ppm test substance in diet
- Group 3: 500 ppm test substance in diet
- Group 4: 1000 ppm test substance in diet
- Group 5: 4000 ppm test substance in diet
- Group 5: 8000 ppm test substance in diet

Observations and examinations performed and frequency:

MORTALITY and CLINICAL SIGNS: Yes
- Time schedule: Twice daily

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed once a week for detailed clinical observations, and six days a week for overt clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight data were collected weekly for all animals.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, food consumption data were collected weekly for all animals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmic examinations were performed prior to treatment and prior to final sacrifice.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Just prior to sacrifice at study termination
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: Yes, overnight
- How many animals: Blood samples were collected from 10 animals/sex/group (or from the surviving animals in the 4000 ppm group) for haematology analyses.
- Parameters checked: Hemoglobin, hematocrit, erythrocyte count, erythrocyte indices, platelet count, total leukocyte count, differential leukocyte count, reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Just prior to sacrifice at study termination
- Animals fasted: Yes, overnight
- How many animals: Blood samples were collected from 10 animals/sex/group for clinical chemistry
- Parameters checked: AST (SGPT), ALT (SGOT), creatinine, alkaline phosphatase, gamma glutamyl transpeptidase, glucose, urea nitrogen, total protein, albumin, globulin, A/G ratio, total bilirubin, direct bilirubin, indirect bilirubin, calcium, phosphorous, sodium, potassium, chloride

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

SACRIFICE: Animals were anesthetized with methoxyflurane and exsanguinated by severing the brachial vessel.
GROSS PATHOLOGY: Yes, all animals (surviving) were subject to gross necropsy.
HISTOPATHOLOGY: Yes, histopathologic examinations were performed on selected tissues from 10 randomly selected animals/group from the control and 1000 ppm dose groups. The 1000 ppm group was selected for complete examination because it was the highest dose group without significant mortality.
- Tissues collected for histopathology: Gross lesions, brain, eyes, pituitary, salivary gland, heart, aorta, thymic region, thyroid, lungs, adrenals, trachea, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, spinal cord, pancreas, liver, kidneys, urinary bladder, testes, prostate, epididymis, ovaries, uterus, spleen, lymph nodes, sciatic nerves, sternum

Other examinations:

ORGAN WEIGHT: Prior to sacrifice, body weights were obtained to allow expression of relative organ weights. The liver, spleen, kidney, heart, brain, adrenals, testes and ovaries were weighed for all animals.

Statistics:

- Parametric variables were compared between the dose and control groups using Levene’s test for homogeneity of variances, by analysis of variance and by pooled variance t-tests.
- Non-parametric data were analyzed by the Kruskal-Wallis test or by the Wilcoxon rank sum test as modified by Mann-Whitney.
- Frequency data were compared using Fisher’s exact tests.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All animals in the 8000 ppm group died. For the 4000 ppm group 12/15 males and 11/15 females died or were sacrificed in a moribund condition.

Mortality:

mortality observed, treatment-related

Description (incidence):

Treatment-related clinical findings were restricted to animals from the 4000 and 8000 ppm groups. The observations were of two types, general cachexia and loose faeces. Findings for the surviving animals were similar to those that died.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Decrease in body weight was observed for the 4000 and 8000 ppm dose group surviving the first week of the study. A slight but significant decrease was also noticed in the males of the 1000 ppm dose group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Decrease in food consumption was observed for the 4000 and 8000 ppm dose group surviving the first week of the study. A slight decrease in the food consumption was also noticed in the males of the 1000 ppm dose group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related finding at any treatment level was observed.

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment related change was observed for males or females from any treatment group (4000 ppm or lower).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment related effect up to 1000 ppm. Significant increases in ALT and phosphorus were observed for 3 surviving males of the 4000 ppm group.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

No treatment related effect up to 1000 ppm was observed.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment related effect up to 1000 ppm was observed. Gross lesions related to the treatment were restricted to the animals that died in the 8000 and 4000 ppm group and to a lesser degree in the animals that survived the 4000 ppm group. The findings were fecal staining of perineal area, emaciation, colour changes of the organs, ileus consisting of distended fluid and gas-filled viscera.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathologic changes were observed for animals in the 4000 and 8000 ppm dose group and consisted of congestion of various organs or tissues, mucosal cell degeneration of the villus tips of small intestine and cecum, submucosal edema of stomach, splenic contraction and hepatocellular atrophy.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOEL

Effect level:

31 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: the dose of 500 ppm is equivalent to 31 mg/kg bw/day for males and 38 mg/kg bw/day for females

Key result

Dose descriptor:

NOEL

Effect level:

38 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: the dose of 500 ppm is equivalent to 31 mg/kg bw/day for males and 38 mg/kg bw/day for females

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

62 mg/kg bw/day (nominal)

System:

other: GI tract, hepatobiliary and spleen (males)

Organ:

colon

ileum

intestine

liver

spleen

stomach

Treatment related:

yes

Dose response relationship:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

77 mg/kg bw/day (nominal)

System:

other: GI tract, hepatobiliary and spleen (females)

Organ:

colon

ileum

intestine

liver

spleen

stomach

Treatment related:

yes

Dose response relationship:

yes

Other than a slight trend in reduced body weight and food consumption in males at 1000 ppm, there were no treatment-related findings at 1000 ppm or less. The highest dose of the test substance lead to 100 and 80% mortality for 8000 and 4000 ppm group respectively indicating 1000 ppm to be the maximal tolerated dose. The animals that survived at 4000 ppm were cachectic and debilitated. The probable cause of death was ileus and shock. The females showed less aberrations in all measurements than the males.

Mortality at high doses was considered to occur at concentrations gastrointestinal mucosa and resulting in dehydration and wasting or affects the gastrointestinal flora. The observed effects were all related to the irritation and corrosivity properties of the substance and no specific organ toxicity was evidenced. The NOEL of the study was based on reduction in body weight and body weight gain, consistent with decreased food consumption. Therefore, it was concluded that all effects could be attributed to local gastrointestinal irritaton/corrosion and consequent reduced food intake without observing any primary systemic effect.

Conclusions:

Based on the results of the read across substance study, the NOAEL for the test substance is considered to be 500 ppm in the diet, i.e., equivalent to 31 mg/kg bw/day (i.e. 25 mg a.i./kg bw/day) for males and 38 mg/kg bw/day (i.e. 30 mg a.i./kg bw/day) for females.

Executive summary:

A study was conducted to determine the repeated dose oral toxicity of the read across substance, C12-16 ADBAC (active ingredient: approx. 80%), according to OECD Guideline 408 and US EPA OPP 82 -1, in compliance with GLP. This sub-chronic oral toxicity study (90 d) was realised in Sprague-Dawley rats. The rats were administered daily dietary levels of 0, 100, 500, 1000, 4000 and 8000 ppm test substance, equivalent to 0, 6, 31 and 62 mg/kg bw/day (i.e. 0, 4.8, 25 and 50 mg a.i./kg bw/day) (males) and 0, 8, 38 and 77 mg/kg bw/day (i.e. 0, 6.4, 30 and 62 mg a.i./kg bw/day) (females) for 95 and 96 days, respectively. Daily intakes at 4000 and 8000 ppm could not be calculated due to high mortality. The animals were observed for mortality, clinical signs, body weight, food consumption, hematology and clinical chemistry at termination. Gross and histopathological examinations were also performed. A slight trend in reduction of body weight and food consumption in males at 1000 ppm was observed. Besides this, there were no treatment-related findings at 1000 ppm or lower. There was 80 and 100% mortality at 4000 and 8000 ppm, respectively. Clinical signs of toxicity (general cachexia and loose faeces), decreased food consumption and body weights, gross necropsy findings (including increased amounts of liquids or semi-solid material within the stomach, jejunum, ileum and cecum) were also observed at 4000 and 8000 ppm. The cause of morbidity and death was assumed to be shock secondary to fluid and/or ionic shifts in the gastro-intestinal tract. Under study conditions, the NOAEL for the C12 -16 ADBAC was considered to be 500 ppm in the diet, i.e., equivalent to 31 mg/kg bw/day (i.e. 25 mg a.i./kg bw/day) for males and 38 mg/kg bw/d (i.e. 30 mg a.i./kg bw/dayS) for females (Van Miller, 1988). Based on the results of the read across substance study, the NOAEL for the test substance, C12 ADBAC, can be considered to be 500 ppm in the diet, i.e., equivalent to 31 mg/kg bw/day (i.e. 25 mg a.i./kg bw/day) for males and 38 mg/kg bw/day (i.e. 30 mg a.i./kg bw/day) for females.

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

skin irritation: in vivo

Type of information:

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

Adequacy of study:

key study

Study period:

1975

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Deviations:

yes

Remarks:

1. In the study, abraded and non abraded skin sites were used; however, the guideline recommends unabraded skin. 2. Exposure period was 24 h; however, guideline recommends 4 h exposure period

GLP compliance:

no

Species:

rabbit

Strain:

not specified

Type of coverage:

occlusive

Preparation of test site:

other: The test substance was applied to both abraded and intact skin

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

Test material
- Amount(s) applied (volume or weight with unit): 0.5 mL

Duration of treatment / exposure:

24h

Observation period:

24 and 72h

Number of animals:

6

Details on study design:

Test site:
- Area of exposure: Back of animal
- Type of wrap if used: The patches consisted of two layers of light gauze cut in squares (2.5 cm on the side). The patches were secured to the test area by thin bands of adhesive tape and the trunk of each animal was wrapped in clear plastic trunk bands after application of the test substance.
- Type of test site: Two areas (intact and abraded) on the back of each animal placed approximately 10 cm apart

Scoring system:
- Skin reactions were scored for erythema and edema, according to Draize Woodland and Calvery scoring system.

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

mean

Time point:

other: 24h and 72h

Score:

6.29

Max. score:

8

Reversibility:

not fully reversible within: 72h

Remarks on result:

other: Intact and abraded skin site

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24h and 72h

Score:

3.33

Max. score:

4

Reversibility:

not fully reversible within: 72h

Remarks on result:

other: Intact skin site

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24h and 72h

Score:

3.5

Max. score:

4

Reversibility:

not fully reversible within: 72h

Remarks on result:

other: Abraded skin site

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24h and 72h

Score:

2.66

Max. score:

3

Reversibility:

not fully reversible within: 72h

Remarks on result:

other: Intact skin site

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24h and 72h

Score:

3

Max. score:

3

Reversibility:

not fully reversible within: 72h

Remarks on result:

other: Abraded skin site

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

other: 24/72 h

Score:

ca. 3.5 - ca. 4

Max. score:

4

Reversibility:

not fully reversible within: 72 h

Remarks on result:

positive indication of irritation

Remarks:

(mean erythema score was: 3.5 for intact and 4 for abraded skin)

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

other: 24 / 72 h

Score:

ca. 3 - ca. 3.5

Max. score:

4

Reversibility:

not fully reversible within: 72 h

Remarks on result:

positive indication of irritation

Remarks:

(mean erythema score was: 3 for intact and 3.5 for abraded skin)

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

other: 24 / 72 h

Score:

ca. 3.5

Max. score:

4

Reversibility:

not fully reversible within: 72 h

Remarks on result:

positive indication of irritation

Remarks:

(mean erythema score was: 3.5 for intact and 3.5 for abraded skin)

Irritation parameter:

erythema score

Basis:

animal #4

Time point:

other: 24 / 72 h

Score:

ca. 3.5

Max. score:

4

Reversibility:

not fully reversible within: 72 h

Remarks on result:

positive indication of irritation

Remarks:

(mean erythema score was: 3.5 for intact and 3.5 for abraded skin)

Irritation parameter:

erythema score

Basis:

animal #5

Time point:

other: 24 / 72 h

Score:

ca. 3

Max. score:

4

Reversibility:

not fully reversible within: 72 h

Remarks on result:

positive indication of irritation

Remarks:

(mean erythema score was: 3 for intact and 3 for abraded skin)

Irritation parameter:

erythema score

Basis:

animal #6

Time point:

other: 24 / 72 h

Score:

ca. 3.5 - ca. 4

Max. score:

4

Reversibility:

not fully reversible within: 72 h

Remarks on result:

positive indication of irritation

Remarks:

(mean erythema score was: 4 for intact and 3.5 for abraded skin)

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: 24 / 72 h

Score:

ca. 2.5 - ca. 3

Max. score:

3

Reversibility:

not fully reversible within: 72 h

Remarks on result:

positive indication of irritation

Remarks:

(mean erythema score was: 2.5 for intact and 3 for abraded skin)

Irritation parameter:

edema score

Basis:

animal #2

Time point:

other: 24 / 72 h

Score:

ca. 2.5 - ca. 3

Max. score:

3

Reversibility:

not fully reversible within: 72 h

Remarks on result:

positive indication of irritation

Remarks:

(mean erythema score was: 2.5 for intact and 3 for abraded skin)

Irritation parameter:

edema score

Basis:

animal #3

Time point:

other: 24/72 h

Score:

ca. 3

Max. score:

3

Reversibility:

not fully reversible within: 72 h

Remarks on result:

positive indication of irritation

Remarks:

(mean erythema score was: 3 for intact and 3 for abraded skin)

Irritation parameter:

edema score

Basis:

animal #4

Time point:

other: 24/72 h

Score:

ca. 2.5 - ca. 3

Max. score:

3

Reversibility:

not fully reversible within: 72 h

Remarks on result:

positive indication of irritation

Remarks:

(mean erythema score was: 2.5 for intact and 3 for abraded skin)

Irritation parameter:

edema score

Basis:

animal #5

Time point:

other: 24/72 h

Score:

ca. 3

Max. score:

3

Reversibility:

not fully reversible within: 72 h

Remarks on result:

positive indication of irritation

Remarks:

(mean erythema score was: 3 for intact and 3 for abraded skin)

Irritation parameter:

edema score

Basis:

animal #6

Time point:

other: 24/72 h

Score:

ca. 2.5 - ca. 3

Max. score:

3

Reversibility:

not fully reversible within: 72 h

Remarks on result:

positive indication of irritation

Remarks:

(mean erythema score was: 2.5 for intact and 3 for abraded skin)

Irritation parameter:

erythema score

Time point:

24/48/72 h

Remarks on result:

other: see below table for details on results

Irritation parameter:

edema score

Time point:

24/48/72 h

Remarks on result:

other: see below table for details on results

Irritant / corrosive response data:

- Severe erythema and edema were observed in animals with abraded and intact skin. See below table for individual animal scores at each time point.

Table 1. Individual animal data on the skin irritation response

Rabbit no.	Reaction	24 h		72 h
		Intact	Abraded	Intact	Abraded
1	Erythema	4	4	3	4
	Edema	2	3	3	3
2	Erythema	3	4	3	3
	Edema	2	3	3	3
3	Erythema	4	4	3	3
	Edema	3	3	3	3
4	Erythema	4	4	3	3
	Edema	2	3	3	3
5	Erythema	3	3	3	3
	Edema	3	3	3	3
6	Erythema	4	4	4	3
	Edema	2	3	3	3

 

Interpretation of results:

other: Category 1B (corrosive) based on CLP criteria

Conclusions:

Based on the results of the read across study, the test substance, C12 ADBAC, can be considered to be corrosive to rabbit skin.

Executive summary:

A study was conducted to determine the skin irritation / corrosion potential of the read across substance, C12 -16 ADBAC (50 -80% active), according to US EPA OPPTS 870.2500. The experiment was performed in rabbits. The undiluted test substance was applied on intact and abraded skin sites using occlusive patches for an exposure period of 24 h. The skin was then observed for erythema and edema formation and the scoring was done according to the Draize, Woodland and Calvery scoring system at 24 and 72 h from the onset of exposure. Severe erythema and edema were observed in all the test animals at both the abraded and intact sites. The mean Primary Irritation Index (PII) of the test substance was calculated to be 6.29 and the mean values of erythema and edema were 3.33 (intact skin site), 3.5 (abraded skin site), 2.66 (intact skin site) and 3 (abraded skin site). Under study conditions, C12 -16 ADBAC is considered to be corrosive to rabbit skin (Wallace, 1975). Based on the results of the read across study, the test substance, C12 ADBAC, can be considered to be corrosive to rabbit skin.