4-poly(propyl), benzene sulfonic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

key study

Study period:

4 November 2008 to 15 April 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: USA EPA Health Effects Test Guidelines OPPTS 870.5050 Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Sponsor's identification: AS305BD
Description: dark brown, viscous liquid
Lot number: TS07002
Analytical purity: 90%
Storage conditions: room temperature in the dark

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: approximately 5-8 weeks old
- Weight at study initiation: 222-299 g (males); 167-218 g (females)
- Fasting period before study:
- Housing: solid floor polypropylene cages
- Diet (e.g. ad libitum): Rodent 2014C Teklad Global Certified Diet, Harlan, Oxon, UK ad libitum
- Water (e.g. ad libitum): mains drinking water ad libitium
- Acclimation period: ten days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 ºC
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least fifteen per hour
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark

Occasional deviations from enevironmental targets were not considered to have affected the purpose or integrity of the study

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Polyethylene glycol 400. Formulations were corrected for 90% purity. The stability and homogeneity of the test material formulations were determined by Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory. Results show the formulations to be stable for four hours. A fresh formulation was therefore made each day and the animals were dosed within four hours of preparation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of test material formulation were taken and analysed for concentration of AS305BD at Harlan Laboratories Ltd, Shardlow, UK Analytical Laboratory. The method used for analysis of formulations and the results obtained are given in Appendix 20. The results indicate that the prepared formulations were within ± 10% of the nominal concentration.

Duration of treatment / exposure:

28 Days

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
60, 250 and 1000 mg/kg/day (incorporating a correction factor for 90% purity).
Basis:
actual ingested

No. of animals per sex per dose:

Three groups, each of five male and five female; Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were chosen based on the results of a previous toxicity study (SPL 0703-0382). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test material, and the results of the study are believed to be of value in predicting the likely toxicity of the test material to man.

- Rationale for animal assignment (if not random):
random

Positive control:

Not applicable.

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
-All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends. During the treatment-free period, animals were observed twice daily, morning and afternoon (once daily at weekends). All observations were recorded.

FUNCTIONAL OBSERVATIONS
Prior to the start of treatment and on Days 7, 14, 21 and 25, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.

Behavioural assessments
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: Gait, Tremors, Twitches, Convulsions, Bizarre/Abnormal/Stereotypic behaviour, Salivation, Pilo-erection, Exophthalmia, Lachrymation, Hyper/Hypothermia, Skin colour, Respiration, Palpebral closure, Urination, Defecation, Transfer arousal, Tail elevation.

Functional performance tests
Motor Activity - twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall hour period and also during the final 20% of the period (considered to be the asymptotic period).

Forelimb/Hindlimb Grip Strength - an automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

Sensory reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed: Grasp response, Vocalisation, Toe pinch, Tail pinch, Finger approach, Touch escape, Pupil reflex, Blink reflex, Startle reflex.

BODY WEIGHT: Yes
- Time schedule for examinations: individual bodyweights were recorded on Day 1 and at weekly intervals thereafter. Bodyweights were also performed prior to terminal kill and, in the case of recovery group animals, on Days 36 and 43 prior to terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: water intake was measured and recorded daily for each cage group.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), Erythrocyte indices (mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC)), Total leucocyte count (WBC), Differential leucocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT), Reticulocyte count (Retic) (Methylene blue stained slides were prepared and reticulocytes were assessed). Prothrombin time (CT) was assessed by 'Innovin' and Activated partial thromboplastin time (APTT) was assessed by 'Actin FS' using samples collected into sodium citrate solution (0.11 mol/I).

CLINICAL CHEMISTRY: Yes
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: Urea, Glucose, Total protein (Tot. Prot.), Albumin, Albumin/Globulin (NG) ratio (by calculation), Sodium (Na+), Potassium (K+), Chloride (CI-), Calcium (Ca++), Inorganic phosphorus (P), Gamma glutamyltranspeptidase (γGT), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Alkaline phosphatase (AP), Creatinine (Creat), Triglycerides (Tri), Total cholesterol (Chol), Total bilirubin (Bili).

URINALYSIS: Yes
The following parameters were measured on collected urine: volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, reducign substances, blood.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 15)

HISTOPATHOLOGY: Yes (see table 16)

Other examinations:

Not applicable

Statistics:

Data were processed to give group mean values and standard deviations where appropriate. All data were summarised in tabular form . Where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett's test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (nonparametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Results and discussion

Results of examinations

Clinical signs:

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

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTLAITY
There were no unscheduled deaths.

CLINICAL OBSERVATIONS
Incidents of increased salivation were detected following dosing for males treated with 1000 mg/kg/day. One male from this treatment group also showed staining around the mouth. An isolated incident of respiratory pattern changes was evident in one female treated with 1000 mg/kg/day during the final week of treatment. No such effects were detected in animals of either sex treated with 250 or 60 mg/kg/day or in recovery 1000 mg/kg/day animals following cessation of treatment (see table 1).

FUNCTIONAL OBSERVATIONS
See Tables 2-5.

Behavioural Assessment.
There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests.
There we re no toxicologically significant changes in the functional performance parameters measured .

Sensory Reactivity Assessments.
There were no treatment-related changes in sensory reactivity.

BODYWEIGHT
No adverse effect on bodyweight development was detected for treated animals (see tables 6-7)

FOOD CONSUMPTION
No adverse effect on dietary intake or food efficiency was detected for treated animals (see tables 8-9).

WATER CONSUMPTION
No intergroup differences were detected (see table 10).

HAEMTOLOGY
Males treated with 1000 mg/kg/day showed statistically significant increases in haemoglobin, erythrocytes, haematocrit and clotting time. No such effects were detected in females treated with 1000 mg/kg/day (see table 11).

BLOOD CHEMISTRY
Animals of either sex treated with 1000 mg/kg/day showed increases in aspartate aminotransferase and alanine aminotransferase together with reductions in cholesterol and triglyceride levels. Males from this treatment group also showed an increase in albumin/globulin ratio. The increase in alanine aminotransferase extended to the 250 mg/kg/day dose level and in females the reduction in cholesterol and triglycerides was also evident. Effects at 60 mg/kg/day were confined to an increase in alanine aminotransferase for females only. No such effects were detected in 60 mg/kg/day males or in recovery animals following fourteen days without treatment.

Recovery 1000 mg/kg/day males showed statistically significant increases in albumin/globulin ratio and phosphorus, and statistically significant reductions in sodium, chloride, aspartate aminotransferase and creatinine whilst recovery 1000 mg/kg/day females showed a statistically significant reduction in albumin following fourteen days without treatment. In the absence of a similar effect seen in nonrecovery animals following the treatment period, the intergroup differences were considered of no toxicological importance. Females treated with 1000 and 250 mg/kg/day showed a statistically significant increase in chloride concentration whilst males treated with 250 mg/kg/day also showed a statistically significant reduction in alkaline phosphatase. In the absence of a true dose-related response the inter group differences were considered of no toxicological significance (see table 12).

URINANALYSIS
No treatment-related effects were seen for treated animals when compared to controls (see table 13).

ORGAN WEIGHTS
No toxicologically significant effects were detected in the organ weights measured (see table 14).

NECROSCOPY
One male treated with 1000 mg/kg/day showed sloughing of the glandular region of the stomach . No such effects were detected in females treated with 1000 mg/kg/day. animals of either sex treated with 250 or 60 mg/kg/day or recovery animals following fourteen days without treatment (see table 15).

HISTOPATHOLOGY
The following treatment-related microscopic changes were detected (see table 16):

Stomach
Minimal to moderate acanthosis and hyperkeratosis were seen in the forestomach of animals of either sex treated with 1000 mg/kg/day. Minimal acanthosis was also seen for males treated with 250 mg/kg/day and one fema le from this treatment level demonstrated minimal hyperkeratosis. Similar effects we re not seen among animals treated at 60 mg/kg/day. Low severity grades of acanthosis , within one instance of associated minimal hyperkeratosis, were seen for two recovery 1000 mg/kg/day females following completion of the recovery period suggesting regression of these conditions. Such epithelial changes in the forestomach of rodents following oral dosing are usually a consequence of an irritant effect of the test material and can be considered adaptive in nature.

Pancreas
Excess accumulation of zymogen granules was seen in exocrine pancreatic cells of animals of either sex treated with 1000 mg/kg/day. Three females treated with 250 mg/kg/day were similarly affected. There was complete regression of the condition among recovery 1000 mg/kg/day animals of either sex.

Mesenteric lymph node
Lymphoid depletion was seen for animals of either sex treated with 1000 mg/kg/day and histiocytosis was seen for three females at this dose level. Similar effects of treatment were not seen for animals of either sex at any other dose level. Althouoh two female recovery 1000 mg/kg/day females demonstrated lymphoid depletion of the mesenteric lymph node, the condition can be regarded as having generally regressed following completion of the recovery period.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Not identified for females.

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

DISCUSSION

The administration of AS305BD by oral gavage, at dose levels of 1000, 250 and 60 mg/kg/day for a period of twenty-eight consecutive days resulted in treatment-related effects in males treated with 1000 and 250 mg/kg/day and at all dose levels for females.

Clinically observable signs were evident for 1000 mg/kg/day animals during the treatment period. Males treated with 1000 mg/kg/day showed increased salivation around the time of dosing and one female from this treatment group showed an isolated incident of respiratory pattern changes. Such observations are commonly observed following the oral administration of an irritant test material formulation . This was further supported by the detection of sloughing of the gastric glandular mucosa at necropsy and the microscopic stomach changes identified as minimal to moderate acanthosis and hyperkeratosis in the forestomach at 1000 mg/kg/day. Minimal acanthosis was also seen for males treated with 250 mg/kg/day and one female from this treatment group demonstrated minimal hyperkeratosis. Such epithelial changes in the forestomach of rodents following oral dosing are usually a consequence of an irritant effect of the test material and can be considered adaptive in nature. Low severity grades of acanthosis, within one instance associated minimal hyperkeratosis, were seen for two recovery 1000 mg/kg/day females following completion of the recovery period thus suggesting partial regression of the condition had occurred.

Haematological investigations revealed increases in haemoglobin, erythrocytes, haematocrit count and clotting time for 1000 mg/kg/day males. Histopathological examinations of the mesenteric lymph nodes revealed lymphoid depletion in animals of either sex treated with 1000 mg/kg/day, with associated histiocytosis in females. Although two female recovery 1000 mg/kg/day females still showed lymphoid depletion the condition can be regarded as having generally regressed following completion of the recovery period.

Blood chemical changes were also evident throughout the treatment groups. Increases in alanine aminotransferase and aspartate aminotransferase were evident at 1000 mg/kg/day together with reductions in cholesterol and triglycerides. Males from this treatment group also showed an increase in albumin/globulin ratio. The reductions in cholesterol and triglycerides extended to 250 mg/kg/day females and the effect on alanine aminotransferase also extended to animals of either sex treated with 250 mg/kg/day and in 60 mg/kg/day females . These metabolic changes would suggest a change in hepatic function , however in the absence of any liver weight changes or any hepatic histological correlates the toxicological significance of these findings is minimal.

Microscopic changes were also evident in the pancreas and examination revealed excess accumulation of zymogen granules in the exocrine pancreatic cells of animals of either sex treated with 1000 mg/kg/day. Three females treated with 250 mg/kg/day were also similarly affected. There was however complete regression of the condition among recovery 1000 mg/kg/day animals following completion of the recovery period.

Due to the effects detected in 60 mg/kg/day females, a 'No Observed Effect Level' for females was not established. The effects detected in 60 mg/kg/day females, in the absence of any histopathological changes, were considered not to represent an adverse health effect as defined by the criteria given in EC labelling guideline of Commission Directive 2001 /59/EC

Applicant's summary and conclusion

Conclusions:

The oral administration of AS305BD to rats for a period of twenty-eight consecutive days at a maximum dose level of 1000 mg/kg/day resulted in treatment-related effects in males treated with 1000 and 250 mg/kg/day and at all dose levels for females. A 'No Observed Effect Level' (NOEL) for females was, therefore not established. Based on the effects detected in 250 mg/kg/day males the 'No Observed Effect Level' (NOEL) and the 'No Observed Adverse Effect Level (NOAEL) for males was considered to be 60 mg/kg/day.

The elevated alanine aminotransferase in 60 mg/kg/day females was considered not to represent an adverse health effect. Based on the effects detected in 250 mg/kg/day females the 'No Observed Adverse Effect Level' (NOAEL) for females was considered to be 60 mg/kg/day.

Executive summary:

In a study performed in line with OCED Guideline 407, the test material was administered by gavage to three groups of five male and five female rats for twenty-eight days at 60, 250 and 1000 mg/kg/day. A control group was treated with vehicle alone. Two recovery groups, each of five males and five females, were treated with high dose or vehicle for twenty-eight days and then maintained without treatment for a further fourteen days. Clinical signs, bodyweight development, food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period.

There were no unscheduled deaths. Clinical Obs ervations. Incidents of increased salivation were detected following dosing for males treated with 1000 mg/kg/day. One male from this treatment group also showed staining around the mouth. An isolated incident of respiratory pattern changes was evident in one female treated with 1000 mg/kg/day during the final week of treatment.

There were no treatment-related changes in the behavioural parameters measured, sensory reactivity, bodyweight, food or water consumption, urinanalysis nor toxicologically significant changes in the functional parameters measured, organ weights.

Males treated with 1000 mg/kg/day showed increases in haemoglobin, erythrocytes, haematocrit and clotting time. No such effects were detected in females treated with 1000 mg/kg/day. Animals of either sex treated with 1000 mg/kg/day showed increases in aspartate aminotransferase and alanine aminotransferase together with reductions in cholesterol and triglyceride levels. Males from th is treatment group also showed an increase in albumin/globulin ratio. The increase in alanine aminotransferase extended to the 250 mg/kg/day dose level and in females the reduction in cholesterol and triglycerides was also evident. Effects at 60 mg/kg/day were confined to an increase in alanine aminotransferase for females only. One male treated with 1000 mg/kg/day showed sloughing of the glandular region of the stomach. No such effects were detected in females treated with 1000 mg/kg/day. Treatment-related microscopic changes were detected in the stomach, pancreas and mesenteric lymph node.

The oral administration of AS305BD to rats for a period of twenty-eight consecutive days at a maximum dose level of 1000 mg/kg/day resulted in treatment-related effects in males treated with 1000 and 250 mg/kg/day and at all dose levels for females. A 'No Observed Effect Level' (NOEL) for females was, therefore not established and was considered to be 60 mg/kg/day for males. The 'No Observed Adverse Effect Level' (NOAEL) was considered to be 60 mg/kg/day for both males and females.