Benzene, 1,1'-oxybis-, tetrapropylene derivs., sulfonated, sodium salts

Endpoint summary

Description of key information

Several GLP and non-GLP studies containing sufficient data for the interpretation and assessment are available, including rat and dog studies on the REACH-registered substance.

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:

1961-1963

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study was conducted prior to GLP and test guidelines, but sufficient data is available for interpretation of results.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Beagle dogs were divided into matched groups and started on diets containing 0.0 (control), 1.0, 0.5, 0.25, or 0.125% Benax 2A1. Four males and four females comprised each group, except for the 1.0% level, which consisted of two males and four females. The percentage levels given above were approximately equivalent to the administration of 0, 319, 128, 65, and 34 mg/kg/day Benax 2A1, respectively, over the two-year period.

General appearance and behavior, growth, food consumption, hematological values, determinations of serum urea nitrogen content, alkaline phosphatase and transaminase activity and bromsulfophthalein dye retention, final organ and body weights, and gross and microscopic examination of the tissues were conducted.

GLP compliance:

no

Remarks:

pre-GLP

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female beagle hounds of approximately three months of age were obtained from a commercial kennel and housed at the Biochemical Research Laboratory. The pups remained in the laboratory three months prior to the beginning of the experiment, during which time they were vaccinated for distemper, hepatitis, and leptospira. The stock diet for the first two months was Famo Labomtory Chow; it was then changed to Purina Laboratory Chow for the remaining time. Dogs of each sex per level were housed together and had free access to food and water at all times.

On October 17, 1961, the dogs were divided into matched groups.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Dogs were divided into matched groups and given diets containing 0.0 (control), 1.0, 0.5, 0.25, or 0.125% Benax 2A1. Four males and four females comprised each group, except for the 1.0% level, which consisted of two males and four females. The percentage levels given above were approximately equivalent to the administration of 0, 319, 128, 65, and 34 mg/kg/day Benax 281, respectively, over the two-year period.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

2 years

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0.0 (control), 1.0, 0.5, 0.25, or 0.125% Benax 2A1
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
0, 319, 128, 65, and 34 mg/kg/day Benax 2A1
Basis:
nominal in diet

No. of animals per sex per dose:

Four males and four females comprised each group, except for the 1.0% level, which consisted of two males and four females.

Control animals:

yes, plain diet

Details on study design:

Beagle dogs were divided into matched groups and started on diets containing 0.0 (control), 1.0, 0.5, 0.25, or 0.125% Benax 2A1. Four males and four females comprised each group, except for the 1.0% level, which consisted of two males and four females. The percentage levels given above were approximately equivalent to the administration of 0, 319, 128, 65, and 34 mg/kg/day Benax 281, respectively, over the two-year period.

Each dog was weighed weekly for the first three months of the experiment and twice a week thereafter. Food consumption was recorded 3 during the first , 12th, and 16th months, and one week out of each month from 18 months to the end of the experiment. Hematological studies and determinations of serum urea nitrogen content and alkaline phosphatase activity were made before the beginning of the experiment and at three, six, nine, 12, 18, and 24 months. Transaminase activity (SGPT) was determined at one and two years and bromsulfophthalein dye retention at one year. Urea nitrogen content and transaminase and alkaline phosphatase activity was determined. Pre-exposure liver biopsies were performed on one dog of each sex per level, as well as at six, 12, and 18 months on the experiment.

At the end of the two-year period the animals were fasted overnight and weighed before examination at autopsy. The lungs, heart, liver, kidney, spleen, testes, and brain were removed and weighed. Portions of each organ,as well as spinal cord, peripheral nerve, pituitary, thyroid, adrenal, aorta, lymph node, thymus, esophagus, stomach, small and large intestine, pancreas, urinary bladder, ovary, uterus, and skeletal muscle were preserved. The tissues were then sent to the International Research and Development Corporation in Mattawan, Michigan, for preparation of hematoxylin-eosin stained sections and microscopic examination.

Positive control:

Non

Observations and examinations performed and frequency:

Each dog was weighed weekly for the first three months of the experiment and twice a week thereafter. Food consumption was recorded 3 during the first , 12th, and 16th months, and one week out of each month from 18 months to the end of the experiment.

Sacrifice and pathology:

Hematological studies and determinations of serum urea nitrogen content and alkaline phosphatase activity were made before the beginning of the experiment and at three, six, nine, 12, 18, and 24 months. Transaminase activity (SGPT) was determined at one and two years and bromsulfophthalein dye retention at one year. Urea nitrogen content and transaminase and alkaline phosphatase activity was determined. Pre-exposure liver biopsies were performed on one dog of each sex per level, as well as at six, 12, and 18 months on the experiment.

At the end of the two-year period the animals were fasted overnight and weighed before examination at autopsy. The lungs, heart, liver, kidney, spleen, testes, and brain were removed and weighed. Portions of each organ,as well as spinal cord, peripheral nerve, pituitary, thyroid, adrenal, aorta, lymph node, thymus, esophagus, stomach, small and large intestine, pancreas, urinary bladder, ovary, uterus, and skeletal muscle were preserved. The tissues were then sent to the International Research and Development Corporation in Mattawan, Michigan, for preparation of hematoxylin-eosin stained sections and microscopic examination.

Other examinations:

None

Statistics:

No data

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Benax 2A1 was fed in the diets of male and female beagle hounds for a period of two years at the concentrations of 1.0, 0.5, 0.25, or 0.125%. Food consumption data indicated that the dietary concentrations given above administered the test substance in amounts of 319, 128, 65 and 34 mg/kg/day respectively. No evidence of adverse effect whatsoever was observed in the dogs given the 0.5% dietary level or below as judged by general appearance and behavior, growth, food consumption, hematological values, determinations of serum urea nitrogen content, alkaline phosphstase and transaminase activity, and bromsulfophthalein dye retention, final body and organ weights, and gross and microscopic examination of the tissue.

The two males and four females which received 1.0% Benax 2A1 in their diets showed growth retardation. Because male dog #330 and female dog #343 lost approximately one-third of their original weights, they were sacrificed after 14 months of the experiment. The final weight of the other male was essentially the same as his original weight, while two of the remaining females lost weight and the third gained. The 1.0% level was not readily acceptable to the dogs. Persistent scratching at the feeders was noted during the early months of the experimental period. This was noted to some extent also in the group of dogs receiving the 0.5% level. Food consumption records for the first month reflect this observation in that the unusually high figures in comparison with the controls was due to spillage and wastage. Therefore, it is possible that the growth retardation in the dogs maintained on the diet containing 1.0% Benax 2A1 may be related somewhat to decreased food intake, at least during the early part of the experiment. Intestinal irritation, as evidenced by loose stools and diarrhea which these dogs exhibited for the first 45 days on the experiment, also may have contributed to their failure to gain weight.

Alkaline phosphatase determinations at various intervals throughout the two-year period showed a slight increase in activity in both of the male dogs and in two of the four females on the 1.0% level. However, the determination of two other liver function tests, that of bromsulfophthalein dye retention at approximately one year and transaminase activity at one and two years, gave normal results in comparison with the control values.

An increased liver/body weight ratio was found in the male dog which was sacrificed after 14 months. The organ/body weight ratio of the kidney of the 1.0% male which was carried through to the end of the two-year period was also increased. However, these variations are due to decreased body weights, since there was no increase in the organ weights when considered on the absolute basis in grams.

Hematological values, serum urea nitrogen determinations, and gross and microscopic examination of the tissues gave no indication of any adverse effects in the dogs which received 1.0% Benax 2A1 in their diets when compared with the controls.

Dose descriptor:

NOAEL

Effect level:

128 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: Based on growth retardation, loose stools and diarrhea, and increased alkaline phosphatase observed in dogs given 319 mg/kg/day or 1.0% in diet. Note: 128 mg/kg/day= 0.5% on diet.

Critical effects observed:

not specified

None

Conclusions:

The systemic NOEL in Beagle dogs was determined to be 0.5% or 128 mg DOWFAX 2A1/kg bw/day.

Executive summary:

Male and female beagle hounds were maintained for two years on diets containing 0.5, 0.25, or 0.125% Benax/DOWFAX 2A1 (equivalent to 128, 65, and 34 mg/kg/day) without evidence of adverse effect as judged by general appearance and behavior, growth, food consumption, hematological values, determinations of serum urea nitrogen content, alkaline phosphatase and transaminase activity and bromsulfophthalein dye retention, final organ and body weights, and gross and microscopic examination of the tissues compared to control animals. At the highest dose level (1% or 319 mg/kg/day, study LOAEL), growth was retarded in the male and female dogs fed Benax/DOWFAX 2A1. These dogs also had loose stools and diarrhea for the first 45 days of the experiment; slightly increased alkaline phosphatase activity in both male dogs and in two out of four female dogs; variations in a few organ/body weight ratios with no difference in organ weights when considered on the absolute basis in grams. Hematological determinations, serum urea nitrogen and transaminase values, determination of bromsulfophthalein dye retention, and gross and microscopic examination of the tissues gave no indication of adverse effects when compared with the controls. Based on these effects, the systemic NOEL in Beagle dogs was determined to be 0.5% or 128 mg DOWFAX 2A1/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

128 mg/kg bw/day

Study duration:

chronic

Species:

dog

Quality of whole database:

acceptable

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

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study was conducted prior to GLP and test guidelines, but sufficient data is available for interpretation of results.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The skin irritation test included topical application of 0.5 ml of neat Dowfax 2A1 to the ear, and to intact and abraded skin on the abdomen of a male New Zealand White (NZW) rabbit. The animal was bandaged for 24 hours after application of the test material to provide occlusion and prevent grooming of the application site. Twenty four hours after application the bandage was removed and the application sites were graded. Five consecutive daily doses of Dowfax 2A1 were applied to the intact abdominal skin, and the apex of the left pinna, and three consecutive daily applications to the abraded abdominal site. Observations were recorded up to 72 hours after the final dose.

GLP compliance:

no

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male

Details on test animals or test system and environmental conditions:

No additional data

Type of coverage:

other: ear- open; intact and abraded abdomen- occluded

Vehicle:

unchanged (no vehicle)

Details on exposure:

The skin irritation test included topical application of 0.5 ml of neat Dowfax 2A1 to the ear, and to intact and abraded skin on the abdomen of a male New Zealand White (NZW) rabbit. The animal was bandaged for 24 hours after application of the test material to provide occlusion and prevent grooming of the application site. Twenty four hours after application the bandage was removed and the application sites were graded

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Five consecutive daily doses of Dowfax 2A1 were applied to the intact abdominal skin, and the apex of the left pinna, and three consecutive daily
applications to the abraded abdominal site.

Remarks:

Doses / Concentrations:
0.5 ml
Basis:
other: amount applied

No. of animals per sex per dose:

1

Control animals:

no

Details on study design:

The skin irritation test included topical application of 0.5 ml of neat Dowfax 2A1 to the ear, and to intact and abraded skin on the abdomen of a male New Zealand White (NZW) rabbit. The abdomen of the rabbit was shaved with a straight razor at least three days prior to test initiation. The animal was
bandaged for 24 hours after application of the test material to provide occlusion and prevent grooming of the application site. Twenty four hours
after application the bandage was removed and the application sites were graded. Five consecutive daily doses of Dowfax 2A1 were applied to the intact abdominal skin, and the apex of the left pinna, and three consecutive daily applications to the abraded abdominal site. The study was terminated 72 hours after the final dose.

Positive control:

None

Observations and examinations performed and frequency:

Twenty four hours after application the bandage was removed and the application sites were graded. Observations were recorded out to 72 hours after the last dose.

Sacrifice and pathology:

No data

Other examinations:

None

Statistics:

None

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

The animal survived the test period. No clinical signs indicative of systemic toxicity were observed.

No irritation was observed on the ear. Very slight erythema was observed at the intact site after the last application. Very slight erythema was observed at the abraded site on test day two through test day five. The study was terminated 72 hours after the final dose.

Critical effects observed:

not specified

None

Conclusions:

No irritation was observed on the ear. Very slight erythema was observed at the intact site after the last application. Very slight erythema was observed at the abraded site on test day two through test day five. The study was terminated 72 hours after the final dose.

Executive summary:

The skin irritation test included topical application of 0.5 ml of neat Dowfax 2A1 to the ear, and to intact and abraded skin on the abdomen of a male New Zealand White (NZW) rabbit. Five consecutive daily doses of Dowfax 2A1 were applied to the intact abdominal skin, and the apex of the left pinna, and three consecutive daily applications to the abraded abdominal site. No irritation was observed on the ear. Very slight erythema was observed at the intact site after the last application. Very slight erythema was observed at the abraded site on test day two through test day five. The study was terminated 72 hours after the final dose.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

5.75 mg/cm²

Study duration:

subacute

Species:

rabbit

Quality of whole database:

adequate

Additional information

ORAL STUDIES OVERVIEW:

2 Year Dog Study:

In the 1963 KS (Klimisch 2), groups of male and female beagle hounds were maintained for two years on diets containing 0, 34, 65, 128 mg/kg/day Benax/DOWFAX 2A1 without evidence of adverse effect as judged by general appearance and behavior, growth, food consumption, hematological values, determinations of serum urea nitrogen content, alkaline phosphatase and transaminase activity and bromsulfophthalein dye retention, final organ and body weights, and gross and microscopic examination of the tissues. Growth was retarded in the male and female dogs fed higest dose of 319 mg/kg/day DOWFAX 2A1 (study LOAEL). These dogs also had loose stools and diarrhea for the first 45 days of the experiment; slightly increased alkaline phosphatase activity in males and half of females; and variations in a few organ/body weight ratios without a difference in organ weights when considered on the absolute basis in grams. Based on all these observations, the general systemic toxicity NOAEL (and point of departure for DNEL derivation) for the dog two year study is 128 mg/kg/day.

 

2 Year Rat Study:

In the 1963 ss (Klimisch 2), groups of 30 male and female rats were maintained for a period of two years on diets containing 0, 15, 50, 150, 500 mg DOWFAX 2A1/kg bw/day without evidence of adverse effect as judged by general appearance and behavior, mortality, incidence of tumorous growths, food consumption, hematological studies, serum urea nitrogen and alkaline phosphatase determinations, bone marrow examination, final average body and organ weights, and gross and microscopic examination of the tissues. Based on depressed growth and statistically significant decrease in the final average body weight at the end of two years seen in female rats fed 500 mg/kg/day BOWFAX 2A1 (study LOAEL), the general systemic toxicity NOAEL for the rat two year study is 150 mg/kg/day.

 

90-Day Rat Study:

In the 1957 ss (Klimisch=2), rats were administered 0, 0.01, 0.03, 0.1, 0.3 and 1.0 % DOWFAX 2A1 in the diet for 90 days. Livers of male rats at the 1% level showed central lobular necrosis of the parenchymal cells, and fatty degeneration and early, but slight fibrosis with some slight bile duct epithelium proliferation in the portal areas when examined microscopically. Female rats at the 1% level showed a retardation of growth together with a statistically significant increase in the average weight of the liver and the kidneys. Grossly, the livers were light in color with a mottled appearance. Upon microscopic examination, effects similar to those noted above in male rats were observed. Based on the above findings the NOEL was 0.3% in the diet.

 

95-Day Dog Study:

In the 1963 ss (Klimisch 2), dogs were administered 0, 40, 131 or 350 mg/kg/day (0, 0.1, 0.3 and 1.0%) DOWFAX 2A1 in the diet for 95 days. The dogs in the 1% or 350 mg/kg/day dose group did not accept their diet to the same degree as the animals on the control or other dietary levels. This decreased food consumption was measured and, on the average, these dogs consumed 73% of the quantity of food consumed by the control group. The corresponding failure to gain weight in the dogs on this level was proportional to the decrease in food consumption in comparison with the controls. Indirectly some organ to body weight ratios were affected in the dogs an this level. However, there was no difference in organ weights when considered on the absolute basis in grams, and the variation in the ratios may be attributed to the decrease in body weight when compared with the controls. There was an increase in the serum alkaline phosphatase values in the dogs which received 1% Benax 2A1 in their diet. However, there were no accompanying pathological changes in the liver nor any significant difference in the liver/body weight ratio when compared with the controls. Based on the above findings the study NOEL was 131 mg/kg/day.

14-Day Rat Study:

Animals were exposed continuously to benzene, 1-1′-oxybis-, tetrapropylene derivs., sulfonated, sodium salts via the diet for 14 consecutive days (Study Days 0-13) at control, 5000, 10,000, and 15,000 ppm. All males and females in the control, 5000, 10,000, and 15,000 ppm groups survived to the scheduled necropsy on Study Day 14. No remarkable clinical observations were noted at any dietary concentration.

Lower mean body weight gains, in the absence of corresponding effects on mean food consumption, were noted during Study Days 0–14 for males at 5000, 10,000, and 15,000 ppm compared to the control group. As a result, mean absolute body weights in the 10,000 and 15,000 ppm group males were 4.1% and 10.0% lower than the control group on Study Day 14. Slightly lower mean body weight gains were also noted for females at 5000 and 10,000 ppm, without corresponding effects on food consumption, throughout the exposure period; however, mean body weights and body weight gains for females in the 15,000 ppm group were generally similar to the control group throughout the exposure period.

At the scheduled necropsy on Study Day 14, dark red areas and/or red discoloration of the lungs were noted for males and females in the 5000, 10,000, and 15,000 ppm groups, as well as the control group. Due to the presence of these findings in all groups, including the control group, and/or the absence of a clear exposure-related response, these findings were considered unrelated to test substance exposure.

Alterations in organ weights noted for males and females at all exposure levels were generally slight, were attributed to a single animal, and/or were not observed in a clear exposure-related manner.

In conclusion, lower mean body weight gains, in the absence of effects on mean food consumption, were noted for males at all dietary concentrations during Study Days 0–14, resulting in mean body weights in the 10,000 and 15,000 ppm group males that were 4.1% and 10.0% lower, respectively, on Study Day 14.

 

Oral Tolerability Study in Rabbits:

The objective of this study was to determine appropriate dietary or dose levels of the test substance, Benzene 1,1’- oxybis-, tetrapropylene derivs., sulfonated, sodium salts, to be evaluated in subsequent developmental toxicity studies in rabbits when administered orally (via gavage or the diet). Animals in Groups 1 and 2 were exposed to the test substance continuously via the diet during Study Days 0–6 and 9–21, with a dosing holiday on Study Days 7 and 8. Animals in Groups 3 and 4 were dosed once daily via oral gavage for 8 and 4 consecutive days, respectively, beginning the second week of exposure for Groups 1 and 2. The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, and food utilization. Oral gavage administration of the test substance was not well tolerated at doses of 250 and 500 mg/kg/day. In the 500 mg/kg/day group, 2 animals were found dead and 1 female was euthanized in extremis on Study Day 3; all 3 females were noted with body weight losses (398 to 496 g; 11.8% to 15.5%) and minimal food consumption (0 to 28 g/day) during Study Days 0–3, with corresponding clinical observations (decreased fecal output, soft feces, and/or thin body) on Study Days 2 and 3. In the 250 mg/kg/day group, all 3 females were noted with body weight losses (274 to 396 g; 8.3% to 11.5%) and minimal food consumption (0 to 64 g/day) during Study Days 0–7, with corresponding excreta-related clinical observations (decreased fecal output and soft feces) during Study Days 3–7. Based on these effects, 250 mg/kg/day was deemed to have exceeded the maximum tolerated dose (MTD), and the females were subsequently euthanized on Study Day 7, thus precluding further evaluation. All animals in the 8000/1000 and 16,000/4000 ppm groups survived to the scheduled euthanasia on Study Day 21. Clinical observations in these groups were limited to red material and red fur staining around the urogenital area for 1 female at 8000 ppm on Study Day 5. Mean body weight losses (123.0 and 243.3 g; 3.7% and 7.2%), with corresponding reduced mean food consumption (36.1 and 26.0 g/day), were noted in the 8000 and 16,000 ppm groups, respectively, during Study Days 0–6. Based on these effects, the animals in these groups were placed on a 2-day dosing holiday (Study Days 7-8), and lower concentrations of 1000 and 4000 ppm were offered for the duration of the study (Study Days 9-21). Following the change in exposure levels, mean body weight gains (135.0 and 139.3 g; 4.1% and 4.3%) and food consumption (114.9 and 97.6 g/day) were noted in the 1000 and 4000 ppm groups, respectively, when Study Days 9-21 were evaluated. No remarkable internal findings were noted for the females found dead, euthanized in extremis, or euthanized due to early group termination. Administration of the test substance via oral gavage at doses of 250 and 500 mg/kg/day to nonpregnant New Zealand White rabbits resulted in severe effects on body weight and food consumption, resulting in mortality/moribundity in the 500 mg/kg/day group on Study Day 3, and subsequent early group termination of the 250 mg/kg/day group on Study Day 7. Administration of the test substance at dietary concentrations of 8000 and 16,000 ppm (equivalent to 11 and 32 mg/kg/day, respectively) for 7 consecutive days (Study Days 0-6) resulted in body weight loss with corresponding reduced food consumption. Based on these effects, the animals in these groups were placed on a 2-day dosing holiday, and concentrations of 1000 and 4000 ppm were offered for the duration of the study (12 consecutive days; Study Days 9-21) and were well tolerated. Based on these findings, follow up developmental toxicity probe in pregnant rabbits will utilize administration of Benzene 1,1’-oxybis-, tetrapropylene derivs., sulfonated, sodium salts at 2000, 4000, and 6000 ppm in the diet.

 

Range-Finding Study in Rats:

 The objective of this study was to determine appropriate dietary levels of the test substance, Benzene 1,1ʹ-oxybis-, tetrapropylene derivs., sulfonated, sodium salts, to be evaluated in a subsequent definitive prenatal developmental toxicity study in rats. Animals were exposed continuously to Benzene 1,1ʹ-oxybis-, tetrapropylene derivs., sulfonated salts continuously via the diet during Gestation Days 6–20 at dose levels of control, 5000, 10,000, and 15,000 ppm. Overt toxicity, including body weight loss, minimal food consumption, and noteworthy clinical observations (erected fur, skin pallor, cold to the touch, and soft feces), were noted at 15,000 ppm, resulting in mortality and subsequent termination of the group prior to the scheduled necropsy. This dietary concentration was therefore considered to have exceeded the maximum tolerated dose. At 10,000 ppm, lower mean body weights, body weight gains, food consumption, and food utilization were noted generally throughout the exposure period, with corresponding clinical observations of soft feces. Lower mean absolute liver and kidney weights, as well as a higher mean litter proportion of post-implantation loss and corresponding lower mean number of live fetuses, were noted compared to the control group. Based on these data, dietary concentrations of below 10,000 ppm were considered appropriate for a definitive prenatal developmental toxicity study of Benzene 1,1ʹ-oxybis-, tetrapropylene derivs., sulfonated, sodium salts administered continuously in the diet to time-mated Crl:CD(SD) rats due to signs of toxicity at the 2 highest concentrations.

Range-Finding Study in Rabbits:

 The objective of this study was to determine appropriate dietary concentrations of the test substance, Benzene 1,1'-oxybis-, tetrapropylene derivs., sulfonated, sodium salts, to be evaluated in a subsequent definitive prenatal developmental toxicity study in rabbits. Animals were exposed to Benzene 1,1'-oxybis-, tetrapropylene derivs., sulfonated, sodium salts, continuously via the diet during Gestation Days 8–28 at dose levels of control, 2000, 4000 and 6000 ppm. All females in the 6000 ppm group were euthanized on Gestation Day 16 due to insufficient test substance administration, as evidenced by markedly reduced food consumption (≤ 50 g/day) for 2-5 consecutive days. Due to the early termination of this group, evaluation of intrauterine survival and external fetal morphology were precluded. An additional female in the 4000 ppm group was euthanized 3 days later (Gestation Day 19) due to insufficient test substance administration, as evidenced by similar markedly reduced food consumption (≤ 40 g/day) for 4 consecutive days. All other females in the control, 2000 and 4000 ppm groups survived to the scheduled necropsy on Gestation Day 29.

Decreased feces was noted for females in the 6000 ppm group at the daily examinations during Gestation Days 13-16, which corresponded to the reduced food consumption noted in this group during this period. No other remarkable clinical observations were noted at any concentration.

Mean maternal body weight losses and lower body weight gains, with corresponding lower mean food consumption, were generally noted for females in the 4000 and 6000 ppm groups from the initiation of exposure on Gestation Day 8 until euthanasia, resulting in a mean body weights that were up to 6.0% (4000 ppm) or 5.4% (6000 ppm) lower than the control group. In the 6000 ppm group, further evaluation of body weight and food consumption data were precluded due to early group termination. In the 4000 ppm group, lower mean adjusted body weight gain and gravid uterine weight were noted for females in this group, while the adjusted body weight was comparable to the control group. A lower mean adjusted body weight gain was noted for females in the 2000 ppm group. Mean body weights, body weight gains, adjusted body weight, and gravid uterine weight in the 2000 ppm group were comparable to the control group.

No remarkable gross macroscopic findings or alterations in mean absolute liver and kidney weights were noted at any concentration.

A higher (19.6% per litter) mean litter proportion of postimplantation loss was noted in the 4000 ppm group compared to the concurrent control group (6.5% per litter), with a corresponding lower (19.9%) mean number of live fetuses (6.3 per dam compared to 7.8 per dam in the control group). These values were also outside of the respective ranges in the Charles River Ashland historical control data for definitive studies. However, given these differences were not statistically significant, were largely attributed to a single litter that consisted of only 1 live fetus and 1 early resorption, and were observed from a small sample size (n=4), the relationship to treatment could not be determined. The mean absolute numbers of early resorptions, late resorptions, and the total number of resorptions in the 4000 ppm group were comparable to the control group. Intrauterine growth in the 2000 ppm group was comparable to the control group.

No external malformations or developmental variations were noted for fetuses in the 2000 and 4000 ppm groups.

In conclusion, due to insufficient test substance administration, as evidenced by markedly reduced mean food consumption for all females at 6000 ppm, and uncertainty around the litter post-implantation loss observation in the 4000 ppm group, dietary concentrations of 4000 ppm and below were selected for a definitive prenatal developmental toxicity study of Benzene 1,1ʹ-oxybis-, tetrapropylene derivs., sulfonated, sodium salts, administered continuously in the diet to time-mated New Zealand White rabbits.

 

OECD 421 Study in Rats:

The objective of this study was to provide information on the potential adverse effects of the test substance, Benzene, 1,1’-oxybis-, tetrapropylene derivs., sulfonated, sodium salts, on male and female reproduction within the scope of a screening OECD 421 study following dietary exposure in rats. This encompassed gonadal function, mating behavior, conception, parturition, and lactation of the parental generation and the development of offspring from conception through day 35 of postnatal life. Animals were exposed to the test substance continuously via the diet. Males were exposed for 14 days prior to mating and continuing through the day of euthanasia (for a minimum of 56 days). Females were exposed for 14 days prior to mating and continuing throughout mating, gestation, and lactation until euthanasia (Lactation Day 21). F1 offspring were potentially exposed in utero and through nursing during lactation. Offspring selected to constitute the  

F1 generation were directly exposed beginning at weaning (Postnatal Day [PND] 21) and continuing until euthanasia (PND 35).  

Dietary test substance concentrations were adjusted based on historical control food consumption and body weight data for lactating females in order to compensate for the higher caloric demands of milk production. Dietary test substance concentrations were also adjusted for F1 pups beginning on PND 21, based on historical control food consumption and body weight data for age-matched animals in order to compensate for rapid growth rates and higher food efficiency during this period.  

The analyzed dietary formulations were within the protocol-specified range of target concentrations and were homogeneous.  

The following parameters and end points were evaluated in this study: mortality, clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen retention, thyroid hormones, macroscopic findings, organ weights, and microscopic examinations, including evaluations of the thyroid gland.

In the 15,000 ppm group, mean body weight losses and lower mean body weight gains, with correspondingly reduced mean food consumption, were noted for F0 males and females beginning immediately following initiation of dietary administration and continuing through approximately 3 weeks of test substance exposure. As a result, mean absolute body weights for F0 males and females in this group were up to 27.5% and 21.8% lower, respectively, than the control group during this period. Therefore, a concentration of 15,000 ppm was considered to have exceeded the maximum tolerated dose, and the group was subsequently terminated and necropsied on 23 Sep 2021 (Study Day 23 [males] or Gestation Day 4, 5, 6, 7, or 8 [females]), thus precluding any further evaluations. No test substance-related macroscopic findings were observed in the 15,000 ppm group at necropsy.

In the 3500 and 7500 ppm groups, there were no test substance-related effects on F0 male or female survival, clinical observations, estrous cyclicity, reproductive parameters, thyroid hormone levels, macroscopic and microscopic findings, or organ weights, including evaluations of the thyroid gland.

Test substance-related lower mean body weight gains, compared to the control group, were noted for F0 males in the 7500 ppm group during the premating period (Study Days 0–14) and when the entire generation (Study Days 0–56) was evaluated, resulting in mean absolute body weights that were 7.9% to 11.1% lower than the control group during Study Days 14–56. These effects were considered adverse. Mean body weights and body weight gains in the 3500 ppm group F0 males were unaffected by test substance exposure.

In F0 females, mean body weights, body weight gains, and food consumption in the 3500 and 7500 ppm groups were unaffected by test substance exposure during the premating period. Lower mean body weight gains, without correlating effects on mean food consumption, were noted in the 3500 and 7500 ppm groups throughout gestation (Days 0–20) compared to the control group, resulting in mean body weights on Gestation Day 20 that were 7.9% and 16.3% lower, respectively, than the control group. The effects on mean body weight during gestation at 7500 ppm were considered test substance-related, and partially attributed to the lower number of pups born in this group, while the lower number of pups born was considered the primary factor in the 3500 ppm group. During lactation (Days 1–21), a higher mean body weight gain was noted in the 7500 ppm group compared to the control group, resulting in a mean absolute body weight on Lactation Day 21 that was comparable to the control group; mean food consumption in this group was comparable to the control group throughout lactation. Mean body weights, body weight gains, and food consumption for F0 females in the 3500 ppm group were unaffected by test substance exposure during lactation.

Lower mean numbers of implantation sites, numbers of pups born, and live litter sizes on PND 0, and higher mean numbers of unaccounted-for sites (indicative of postimplantation loss) were noted at 3500 and 7500 ppm compared to the control group. In addition, lower mean postnatal survival was noted in these same groups during birth to PND 4 compared to the control group, which were attributed to 1 dam in each group with total litter loss on Lactation Day 0. These differences were considered test substance-related and adverse. Mean postnatal survival in these groups was unaffected by the test substance during PND 4–21. No other test substance-related findings were noted in the F1 pups prior to weaning. In the 7500 ppm group, 6(2) pups (litters) were noted with missing eyes bilaterally; however, anophthalmia is a common external malformation in the current OECD 414 historical control data. Mean body weights, body weight gains, anogenital distances (absolute and relative to cube root of bodyweight), areolae/nipple anlagen, thyroid hormone levels on PND 21, macroscopic findings, and organ weights in F1 pups were unaffected by parental exposure to the test substance in the 3500 and 7500 ppm groups.

F1 survival, clinical observations (with the exception of the previously mentioned missing eyes), body weights, body weight gains, food consumption, macroscopic findings, and organ weights were unaffected by direct exposure to the test substance at 3500 and 7500 ppm during PND 21-35.

In conclusion, under the conditions of this OECD 421 screening study, a concentration of 7500 ppm (equivalent to 446 to 506 mg/kg/day for males and females) was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity of Benzene 1,1’-oxybis-, tetrapropylene derivs., sulfonated, sodium salts when administered continually in the diet to Crl:CD(SD) rats. The NOAEL for F0 systemic toxicity was considered to be 3500 ppm (equivalent to 193 to 258 mg/kg/day for males and females), based on effects on body weight for F0 males and females at 7500 ppm and excessive toxicity (early group termination) noted at 15,000 ppm. The NOAEL for F1 neonatal toxicity was unable to be determined based on the lower numbers of pups born and lower postnatal survival from birth to PND 4 in both treated groups (3500 and 7500 ppm); some of these effects were driven by a single dam/group and need to be followed up in the definitive OECD 443 study. Based on the lack of any evidence of toxicity, the NOAEL for F1 systemic toxicity was considered to be 7500 ppm (equivalent to 427 and 483 mg/kg/day for males and females, respectively).

DERMAL:

In the 1993 (Klimisch 2) subacute study, the skin irritation test included topical application of 0.5 ml of neat DOWFAX 2A1 to the ear, and to intact and abraded skin on the abdomen of a male New Zealand White rabbit. Five consecutive daily doses of DOWFAX 2A1 were applied to the intact abdominal skin, and the apex of the left pinna, and three consecutive daily applications to the abraded abdominal site. No irritation was observed on the ear. Very slight erythema was observed at the intact site after the last application. Very slight erythema was observed at the abraded site on test day two through test day five. The study was terminated 72 hours after the final dose.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Longest repeat dose study with DOWFAX 2A1 with lowest NOAEL. All durations of exposure to test material showing similar effects in test animals.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
sufficient data available on the registered substance for assessment.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
sufficient data available on the registered substance for assessment.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

Justification for classification or non-classification

Non-classification based on the chronic NOAEL of 128 mg/kg/day in accordance with the CLP.