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REACH

2,2'-(2-methylpropylidene)bis[4,6-xylenol]

EC number: 251-394-8 | CAS number: 33145-10-7

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

Repeated dose toxicity: oral

28-day NOAEL: 10 mg/kg/day (based on clinical signs and behavioural changes observed in the functional observations tests).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 June 2016 to 30 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Organisation of Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2015, with the exception of the procedures described in the section litter size.

Deviations:

yes

Remarks:

See "any other information" for details

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650

Version / remarks:

The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

yes

Remarks:

see "Any other information" for details

Principles of method if other than guideline:

In addition, the procedures described in the report essentially conform to the following guidelines:
OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 2015.
The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.
OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

GLP compliance:

yes

Limit test:

Specific details on test material used for the study:

No further details specified in the study report.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Test system: Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and non-pregnant females and untreated animals were used at initiation of the study.
Rationale: This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies.
Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test system: Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and non-pregnant females and untreated animals were used at initiation of the study.
Source F0: Charles River Deutschland, Sulzfeld, Germany.
Age at start pretest: Females: approximately 10-12 weeks.
Age at start F0-treatment: Males: approximately 10-12 weeks. Females: approximately 12-14 weeks.
Number of F0-animals: 48 females and 40 males.
At the end of the pretest phase, 40 females with at least two regular estrous cycles were selected at random and continued in the study. The remaining females were removed from the study.
Acclimatization F0: At least 5 days prior to start of pretest (females) or treatment (males).
Health inspection F0: At least upon receipt of the animals.
Randomization F0: Before initiation of pretest, by computer-generated random g algorithm according to body weight, with all animals within ± 20% of the sex mean.
Identification F0: During pretest (females only): by indelible marker, numbered 101 through 148 at random. During treatment (males and females): by earmark and tattoo.

Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 room air changes/hour, and a 12-hour light/12-hour dark cycle: the photoperiod was between 07:00 and 19:00 hrs daily. The light/dark cycle was interrupted for study related activities. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Accommodation
Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
General: Sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
Water: Free access to tap-water. During motor activity measurements, animals did not have access to water for a maximum of 2 hours.
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.

Vehicle:

propylene glycol

Details on oral exposure:

Vehicle: Propylene glycol, specific gravity 1.036 (Merck, Darmstadt, Germany).
Rationale for vehicle: Based on trial formulations performed at Charles River Den Bosch.
Method of formulation: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle.
Appearance of formulations: Solution (Groups 2-4).
Storage conditions: At room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dose formulations taken on two occasions during the treatment phase, i.e. on 08 Augustus (week 1) and on 06 September 2016 (week 5 of study), were analysed according to a validated method (Test Facility Study no. 512752, ABL Project 16101).
Samples taken in week 1 were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
Samples taken in week 5 were from the Group 2 formulation only and were analysed for determination of stability (over 5 hours at room temperature) and confirmation of accuracy, homogeneity, because the concentration of 0.6 mg test item/ml in this Group 2 formulation was outside the range of 1 – 200 mg/mL checked in the validated method.

Duration of treatment / exposure:

Males were exposed for 31 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 49-56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were exposed for 40-42 days.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Dose / conc.:

3 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

No. of animals per sex per dose:

One control group and three treated groups were tested, each consisting of 10 males and 10 females.

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose levels
Based on the results of a 10-day dose range finder, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 3, 10 and 30 mg/kg.

Positive control:

Not required

Observations and examinations performed and frequency:

Mortality / Viability: At least twice daily.

Clinical signs: At least once daily from start of treatment onwards up to the day prior to necropsy detailed clinical observations were made for all animals, at least 1.5 hours (± 30 min) after treatment (on the peak period of anticipated effects after treatment). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Functional Observations The following functional observations tests were performed on each individual animal of the selected 5 animals/sex/group:
-hearing ability (HEARING), pupillary reflex (PUPIL L/R), and static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
-fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
-locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA).
Total movements and ambulations are reported.
Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.
The selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13). These tests were performed starting after the observation for clinical signs (incl. arena observation, if applicable) at 1.5 hours (±30 min) after treatment.

Body weights: Males and females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on
Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

Clinical Laboratory Investigations (F0-Generation only)
Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m.
The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with K3-EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids. The following parameters were determined:

Haematology
The following haematology parameters were determined in blood prepared with K3-EDTA as an anti-coagulant, using the ADVIA® 2120i Hematology System (Siemens Healthcare Diagnostics B.V., Den Haag, The Netherlands):
White blood cells (WBC); Red blood cells; Reticulocytes; Red blood cell distribution width (RDW); Haemoglobin; Haematocrit; Mean corpuscular volume (MCV); Mean corpuscular haemoglobin (MCH); Mean corpuscular haemoglobin concentration (MCHC); Platelets. Differential leucocyte count: Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils

The following clotting parameters were determined in plasma prepared with citrate as anticoagulant, using the STA Compact® (Diagnostica Stago S.A.S., Asnières, France):
Prothrombin Time (PT); Activated Partial Thromboplastin Time (APTT)

Clinical Biochemistry
The following clinical biochemistry parameters were determined using the AU400 (Beckman Coulter Nederland B.V., Woerden, The Netherlands). All parameters were determined in plasma, except for bile acids which were determined in serum:
Alanine aminotransferase (ALAT); Aspartate aminotransferase (ASAT); Alkaline Phosphatase (ALP); Total protein; Albumin; Total Bilirubin; Bile acids; Urea; Creatinine; Glucose; Cholesterol; Sodium; Potassium; Chloride; Calcium; Inorganic Phosphate (Inorg. Phos).

Sacrifice and pathology:

F0-generation - Termination
The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available.
All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.
Necropsy was conducted on the following days:
Condition Day of necropsy
Males Following completion of the mating period (a minimum of 28 days of dose administration).
Females which delivered PND 14-16.
Females which failed to deliver Post-coitum Days 25-27 (females with evidence of mating)
(nos. 62 and 67)

F0-generation – Macroscopic Examination
After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
The number of former implantation sites were recorded for all paired females.
Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).

Selected 5 animals/sex/group (see Allocation):
Identification marks: not processed; (Aorta); Brain – cerebellum, mid-brain, cortex (7-levels); Caecum; Cervix; Clitoral gland; Colon; Coagulation gland; (Cowper’s gland); Duodenum; Epididymides; Eyes (with optic nerve (if detectable) and Harderian gland); Mammary gland area (males and females); Femur including joint; (Glans penis); (Levator ani plus bulbocavernous muscle complex (LABC)); Heart; Ileum; Jejunum; Kidneys; (Lacrimal gland, exorbital); (Larynx); Liver; Lung, infused with formalin; Lymph nodes – mandibular, mesenteric; (Nasopharynx); (Esohagus); Ovaries; (Pancreas); Peyer’s patches [jejumum, ileum] if detectable; Pituitary gland; Preputial gland; Prostate gland; Rectum; (Salivary glands – mandibular, sublingual); Sciatic nerve; Seminal vesicles; Skeletal muscle; (Skin); Spinal cord – cervical, midthroatic, lumbar; Spleen; Sternum with bone marrow; Stomach; Testes; Thymus; Thyroid including parathyroid if detectable; (Tongue); Trachea; Urinary bladder; Uterus; Vagina; All gross lesions.
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

All remaining animals, males that failed to sire, females which failed to deliver and females with total litter loss:
Cervix; Clitoral gland; Coagulation gland; Cowper’s glands; Epididymides; Glans penis; Levator ani plus bulbocavernosus muscle complex (LABC); Mammary gland area (males and females); Ovaries; Preputial gland; Prostate gland; Seminal vesicles; Testes; Thyroid including parathyroid if detectable; Uterus; Vagina; All gross lesions; Identification marks: not processed

Reproductive organs were only examined by the pathologist for males that failed to sire and all females that failed to deliver healthy pups. Female mammary gland area was not examined by the pathologist as there were no females with total litter loss.

F0-generation – Organ Weights
The following organ weights and terminal body weight were recorded from the following animals:

Selected 5 animals/sex/group (see Allocation):
Adrenal glands; Brain; Cowper’s glands; Epididymides; Glans penis; Heart; Kidneys; Levator ani plus bulbocavernosus muscle complex (LABC); Liver; Ovaries; Prostate; Seminal vesicles including coagulating glands; Spleen; Testes; Thymus; Thyroid (including parathyroid if detectable); Uterus (including cervix)
All remaining animals:
Cowper’s glands; Epididymides; Glans penis; Levator ani plus bulbocavernosus muscle complex (LABC); Testes; Thyroid
Absolute organ weights and organ to body weight ratios are reported.

F0-generation - Histotechnology
All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

F0-generation – Histopathology
The following slides were examined by a pathologist:
The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4 (see Allocation).
Additional slides of the testes of the selected 5 males (see Allocation) of Groups 1 and 4 and all males that failed to sire (see table below) to examine staging of spermatogenesis.
All gross lesions of all animals (all dose groups).
Thyroid gland and liver of all selected 5 males of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups.
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
A peer review on the histopathology data was performed by a second pathologist.

Other examinations:

Further examinations relating to reproduction are details in Section 7.8.1.

Statistics:

The following statistical methods were used to analyse the data:
If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
The Steel-test (Ref. 3; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
The Fisher Exact-test (Ref. 4) was applied to frequency data.
The Kruskal-Wallis nonparametric ANOVA test (Ref. 5) was applied to motor activity data to determine intergroup differences.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Within one week after start of treatment, uncoordinated movements were observed in a few males and females treated at 30 mg/kg/day at 1-2 hours after dosing. The appearance of uncoordinated movements was slightly earlier in females than in males, i.e on day 4 and on day 6 after start of treatment, respectively. The number of males and females affected gradually increased during the treatment-period until the majority of animals showed uncoordinated movements after three weeks of treatment. The presence of this symptom was not consistent in most of the animals or was observed only incidentally. Moreover, in females the degree of uncoordinated movements varied between 1 and 2 (maximum possible grade 3) in several animals, whereas in males uncoordinated movements grade 1 only were observed.
In addition, in some females temporary behavioural changes, including lethargy, restlessness or fearfulness, were observed at the end of the post-coitum or early in the lactation phase.
Salivation was observed for a large part of the study immediately after treatment. However, on-line recording of the observation of salivation was performed from week 4 of treatment onwards. Salivation was observed immediately after dosing in a dose related manner among the animals of all test groups. Salivation was not observed in any of the animals at the observation performed at the time of peak effect 1-2 hours after dosing. The number of males per dose group exhibiting salivation was slightly higher than the number of females of the concurrent dose group. Salivation was also observed in a single male and female in the vehicle control group. Salivation was considered to be a physiological response to the formulation rather than a sign of systemic toxicity considering the nature of the effect and its time of occurrence (i.e. after dosing).
Piloerection was observed in one female of the mid- and one of the high-dose group for a short period shortly after delivery. Signs of discomfort in females at the time of delivery are occasionally observed and in this case, also based on the single occurrences, not related to treatment.
Incidental findings that were noted included, rales and scabs or alopecia in various locations.
These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study.
On one occasion (Day 13 of treatment), some fluid was observed in the oral cavity of female 55 during dosing, likely originated from the stomach and might have contained test formulation, stomach contents and/or saliva. At the incidence observed, these incidental findings were considered not to be signs of toxicological relevance.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

For males and females, body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
The body weight gain in females treated at 30 mg/kg/day on day 4 post coitum (PC), when compared to day 0 PC, was slightly lower and reached a level of statistical significance when compared to the body weight gain in controls. Since body weight gain in these high dose females from day 7 PC onwards was similar to that in controls and the other test groups, the difference in body weight on day 4 PC was considered of no toxicological significance.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption before or after allowance for body weight was similar between treated and control animals.
In high dose females, the food consumption relative to body weight was slightly lower over the last few days of the post-coitum phase (days 17-20) and over the lactation phase, achieving a level of statistical significance at days 17-20 post-coitum when compared to that in controls. The lower values for relative food consumption were the result of slightly higher body weights in combination with a normal food consumption in high dose females. These changes in high dose females might be explained by the assumption that the test item had some nutritional value. However, no toxicological relevance was attached to these findings.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological parameters of treated male and female rats were considered not to be affected by treatment.
In the absence of a treatment related distribution, the levels of statistical significance observed for the coagulation parameter PT in the low and mid dose males were considered not to be of toxicological relevance. Moreover, (minimal) shortening of the PT is considered to have little clinical relevance.
The statistically significant changes in mid dose females for monocytes and haematocrit were considered not to be toxicologically relevant as they occurred in the absence of a treatment related distribution. Moreover, the individual monocytes and haematocrit values in these females remained within the range considered normal for female rats of this age and strain.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical biochemistry parameters of treated male and female rats were considered not to be affected by treatment.
The statistically significant changes for chloride (mid dose males) and total protein (mid dose females), when compared to controls, were considered not to be toxicologically significant as they occurred in the absence of a treatment-related distribution. Moreover, no corroborative findings were observed in these animals and all values were within normal limits.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex, and static righting reflex were normal in all selected animals.
A dose-related decrease in grip strength of the fore limbs was observed in both males and females, achieving a level of statistical significance in the males treated at 30 mg/kg/day compared to the concurrent vehicle controls. The grip strength of the hind limbs was similar in all dose groups and comparable to that in controls in males as well as in females.
In the motor activity test, comprising measurement of movements and ambulations in 12 successive intervals of 5 minutes each, all male groups showed a similar habituation profile with very high activity in the first interval that gradually decreased over the duration of the test period.
However, in one male treated at 3 mg/kg/day (no. 14) an aberrant habituation pattern was observed. Relatively high movements and ambulations were recorded over the 3rd to 7th time interval for this male, resulting in high total values for these parameters of more than twice the average of the other four males of this group. Exclusion of male no 14 from the group resulted in similar group mean values for movements and ambulation compared to the other groups. The aberrant habituation pattern in this male was considered a fortuitous finding and not related to treatment.
In females, the habituation pattern in the controls and low and mid dose were similar to that observed in males, but a different habituation pattern was observed in high dose females, receiving 30 mg/kg bw/day. After a normal, high activity in these high dose females in the first interval, the activity in the second interval was immediately decreased to a (very) low level, for both total movements and ambulations, and remained low until the end of the test.
One high dose female (no. 71), however, showed an increase in activity in the 3rd interval which remained relatively high over the complete test period. As a result this female showed the highest activity of all females in study. Exclusion of female no 71 from the group resulted in high dose group mean values for total movements and ambulations that were clearly lower than the other groups and a standard deviation of the group mean that was comparable to those of the other group means. On the other hand, as the effects on motor activity in the high dose females were suspected to be treatment-related, the high variation in total number of movements and total number of ambulations among the individual females of the high dose group might be the result and do not legitimate exclusion of any female from the high dose group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

A small increase in mean liver weight was observed in males treated at 30 mg/kg/day, achieving a level of statistical significance for the liver/body weight ratio when compared to controls.
Increased epididymides weights were observed in low and high dose males, achieving levels of statistical significance for both the absolute and relative epididymides weights in low and high dose males and for relative weights in high dose males only when compared to controls.
In the absence of a clear dose response relationship and since no histopathological changes were observed in the epididymides and no effects on reproduction data were apparent in the low and high dose females and its litters, the small increases in epididymides weights were considered not related to treatment and of no toxicological significance. Moreover, all values for epididymides weights were within normal limits
In female rats, decreased mean uterus weights were noted, achieving a level of statistical significance for the absolute weights in the high dose females treated at 30 mg/kg/day and for the relative weights in the mid and high dose females treated at 10 and 30 mg/kg/day, respectively, when compared to controls. The statistical significances might be the result of relative high uterus weights observed in control females, and since all individual uterus weights of females in all groups remained within the range considered normal for female rats of this age and strain, no clear dose response relationship was observed, at least for the relative uterus weights, and no histopathological changes were found in the uterus, no toxicological significance was attached to this finding.
The statistically significant difference between relative brain weights of mid dose treated and control females was considered not to be a sign of toxicity in the absence of a dose response relationship.
All other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment with Lowinox® 22IB46.
The incidence of macroscopic findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose effect relationship. These macroscopic findings were therefore considered not to be of toxicological relevance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with LOWINOX® 22IB46 were noted in the liver and thyroid gland of the 30 mg/kg/day group males.
Liver: Hepatocellular hypertrophy was recorded in the liver of all males at 30 mg/kg/day at minimal degree.
Thyroid gland: An increased incidence and severity of follicular cell hypertrophy, up to a slight degree, was present in males treated at 30 mg/kg/day.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

Lowinox® 22IB46 was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 3, 10 and 30 mg/kg. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 31 days). The females that delivered were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 49-56 days). Females that failed to deliver healthy offspring were exposed for 40-42 days.
Based on the results of formulation analysis it was considered that the formulations were prepared accurately and homogenously, and were stable for at least 5 hours at room temperature down to concentrations as low as 0.6 mg/mL (equivalent to a dose level of 3 mg/kg).
Parental results:
Uncoordinated movements were observed for a major part of the study period in both high dose males and females treated at 30 mg/kg/day. In some of these females, temporary behavioural changes, including lethargy, restlessness or fearfulness, were occasionally observed. The functional observation test performed towards the end of the treatment period showed decreased fore limb grip strength in both high dose males and females and an accelerated decrease in movements and ambulations in the motor activity test in high dose females only in contrast to a more gradual decrease in activity in a normal habituation pattern. There might be a relation between the clinical signs and behavioural changes which might be interpreted as signs of neurotoxicity caused by treatment with LOWINOX® 22IB46 at 30 mg/kg/day. In the absence of changes in gait/motility and effects on motor activity in the mid dose animals, the minimal decreases in fore limb grip strength observed at this dose level were considered to be non-adverse and of no toxicological significance.
Small increases in liver weights were observed in high dose males. Histopathology of the liver revealed minimal hepatocellular hypertrophy, but in the absence of any degenerative findings this was considered to be a non-adverse finding.
Furthermore, histopathology also revealed follicular cell hypertrophy of the thyroid gland in males at 30 mg/kg/day. The minor increase in incidence and/or severity (up to slight degree) and the fact that it is usually an adaptive response to induction of hepatic enzymes, this finding was regarded to be an adaptive change and considered to be non-adverse.
No treatment-related and/or toxicologically significant changes were noted in any of the other parental parameters investigated in this study (i.e. body weight, food consumption, clinical laboratory investigations and macroscopic examination).

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

clinical signs

histopathology: non-neoplastic

Key result

Critical effects observed:

Lowest effective dose / conc.:

30 mg/kg bw/day (nominal)

System:

autonomic nervous system

Organ:

other: Not organ specific - affected movement & grip strength

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Functional Observations

Total movements Group 4:

Total Movements	Group 4
Total Movements	All (5) females	Excl. no. 71
Mean	1496	1043
St. dev.	1096	481

Microscopic Examination

Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals

Males

Does level (mg/kg/day):

Liver^a

Hepatocellular hypertrophy

Minimal

Thyroid gland^a

Hypertrophy follicular cell

Minimal

Slight

^a= Number of tissues examined form each group

CLINICAL SIGNS SUMMARY

MALES

PRE MATING

SIGN (MAX. GRADE)

(LOCATION)

WEEK:

DAY:

GROUP 1 (CONTROL)

Secretion / excretion

Salivation (3)

GROUP 2 (3 MG/KG)

Breathing

Rales (3)

Skin / fur

Scabs (3)

(Back)

Secretion / excretion

Salivation (3)

%:

G:
%:

G:
%:

GROUP 3 (10 MG/KG)

Secretion / excretion

Salivation (3)

G:
%:

GROUP 4 (30 MG/KG)

Gait / motility

Uncoordinated movements (3)

Breathing

Rales (3)

Skin / fur

Scabs (3)

(Back)

Scabs (3)

(Tail)

Secretion / excretion

Salivation (3)

G:
%:
G:
%:

G:
%:

MALES

MATING PERIOD

SIGN (MAX. GRADE)

(LOCATION)

WEEK:

DAY:

GROUP 1 (CONTROL)

Secretion / excretion

Salivation (3)

GROUP 2 (3 MG/KG)

Breathing

Rales (3)

Skin / fur

Scabs (3)

(Back)

Secretion / excretion

Salivation (3)

%:

G:
%:

G:
%:

GROUP 3 (10 MG/KG)

Secretion / excretion

Salivation (3)

G:
%:

GROUP 4 (30 MG/KG)

Gait / motility

Uncoordinated movements (3)

Breathing

Rales (3)

Skin / fur

Scabs (3)

(Back)

Scabs (3)

(Tail)

Secretion / excretion

Salivation (3)

G:
%:
G:
%:

G:
%:

G: Median value of the highest individual daily grades

%: Percent of affected animals (0=less than 5%, 1=between 5% and 15%, …., A=more than 95%)

.: Observation performed, sign not present.

CLINICAL SIGNS SUMMARY

FEMALES

PRE MATING

SIGN (MAX. GRADE)

(LOCATION)

WEEK:

DAY:

GROUP 1 (CONTROL)

Breathing

Rales (3)

Skin / fur

Alopecia (3)

Secretion / excretion

Salivation (3)

G:
%:

G:
%:

GROUP 2 (3 MG/KG)

Skin / fur

Alopecia (3)

Secretion / excretion

Salivation (3)

G:
%:

G:
%:

GROUP 3 (10 MG/KG)

Breathing

Rales (3)

Skin / fur

Piloerection (1)

Alopecia (3)

Secretion / excretion

Salivation (3)

G:
%:

GROUP 4 (30 MG/KG)

Behaviour

Restless (3)

Fearful (3)

Lethargy (3)

Gait / motility

Uncoordinated movements (3)

Breathing

Rales (3)

Skin / fur

Piloerection (1)

Alopecia (3)

Secretion / excretion

Salivation (3)

G:
%:
G:
%:
G:
%:

G:
%:

G:
%:
G:
%:

G:
%

FEMALES

MATING PERIOD

SIGN (MAX. GRADE)

(LOCATION)

WEEK:

DAY:

GROUP 1 (CONTROL)

Breathing

Rales (3)

Skin / fur

Alopecia (3)

Secretion / excretion

Salivation (3)

G:
%:

G:
%:

GROUP 2 (3 MG/KG)

Skin / fur

Alopecia (3)

Secretion / excretion

Salivation (3)

G:
%:

G:
%:

GROUP 3 (10 MG/KG)

Breathing

Rales (3)

Skin / fur

Piloerection (1)

Alopecia (3)

Secretion / excretion

Salivation (3)

G:
%:

GROUP 4 (30 MG/KG)

Behaviour

Restless (3)

Fearful (3)

Lethargy (3)

Gait / motility

Uncoordinated movements (3)

Breathing

Rales (3)

Skin / fur

Piloerection (1)

Alopecia (3)

Secretion / excretion

Salivation (3)

G:
%:
G:
%:
G:
%:

G:
%:

G:
%:
G:
%:

G:
%

FEMALES

MATING PERIOD

SIGN (MAX. GRADE)

(LOCATION)

WEEK:

DAY:

GROUP 1 (CONTROL)

Breathing

Rales (3)

Skin / fur

Alopecia (3)

Secretion / excretion

Salivation (3)

G:
%:

G:
%:

GROUP 2 (3 MG/KG)

Skin / fur

Alopecia (3)

Secretion / excretion

Salivation (3)

G:
%:

G:
%:

GROUP 3 (10 MG/KG)

Breathing

Rales (3)

Skin / fur

Piloerection (1)

Alopecia (3)

Secretion / excretion

Salivation (3)

G:
%:

GROUP 4 (30 MG/KG)

Behaviour

Restless (3)

Fearful (3)

Lethargy (3)

Gait / motility

Uncoordinated movements (3)

Breathing

Rales (3)

Skin / fur

Piloerection (1)

Alopecia (3)

Secretion / excretion

Salivation (3)

G:
%:
G:
%:
G:
%:

G:
%:

G:
%:
G:
%:

G:
%

G: Median value of the highest individual daily grades

%: Percent of affected animals (0=less than 5%, 1=between 5% and 15%, …., A=more than 95%)

.: Observation performed, sign not present.

FUNCTIONAL OBSERVATIONS SUMMARY

MALES

GROUP 1

CONTROL

GROUP 2

3 MG/KG

GROUP 3

10 MG/KG

GROUP 4

30 MG/KG

AT WEEK 4

HEARING

SCORE 0/1

MEDIAN

PUPIL L

SCORE 0/1

MEDIAN

PUPIL R

SCORE 0/1

MEDIAN

STATIC R

SCORE 0/1

MEDIAN

GRIP FORE

GRAM

MEAN

ST. DEV.

1299

171

1223

130

1185

163

996*

168

GRIP HIND

GRAM

MEAN

ST. DEV.

570

565

558

174

557

FEMALES

GROUP 1

CONTROL

GROUP 2

3 MG/KG

GROUP 3

10 MG/KG

GROUP 4

30 MG/KG

AT LACTATION

HEARING

SCORE 0/1

MEDIAN

PUPIL L

SCORE 0/1

MEDIAN

PUPIL R

SCORE 0/1

MEDIAN

STATIC R

SCORE 0/1

MEDIAN

GRIP FORE

GRAM

MEAN

ST. DEV.

1113

298

1091

129

968

146

917

GRIP HIND

GRAM

MEAN

ST. DEV.

500

536

126

590

531

113

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

+/++ Steel-test significant at 5% (+) or 1% (++) level

MOTOR ACTIVITY TEST SUMMARY

MALES

GROUP 1

CONTROL

GROUP 2

3 MG/KG

GROUP 3

10 MG/KG

GROUP 4

30 MG/KG

AT WEEK 4

Total Movements

MEAN¹

STD. DEV

2814

280

3457

1657

2754

1195

2442

618

Ambulations

MEAN¹

STD. DEV

519

144

668

316

541

160

487

FEMALES

GROUP 1

CONTROL

GROUP 2

3 MG/KG

GROUP 3

10 MG/KG

GROUP 4

30 MG/KG

AT LACTATION

Total Movements

MEAN¹

STD. DEV

1983

565

1977

410

1960

503

1496

1096

Ambulations

MEAN¹

STD. DEV

442

151

469

142

557

197

470

280

*/** Wilcoxon test significant at 5% (*) or 1% (**) level

¹Group mean of al intervals combined

BODY WEIGHTS (GRAM) SUMMARY

MALES

GROUP 1

CONTROL

GROUP 2

3 MG/KG

GROUP 3

10 MG/KG

GROUP 4

30 MG/KG

PRE MATING

DAY 1

WEEK 1

MEAN

ST. DEV

322

12.3

331

8.4

321

8.5

322

5.7

DAY 8

WEEK 2

MEAN

ST. DEV

341

13.3

348

10.9

340

9.1

340

6.1

MATING PERIOD

DAY 1

WEEK 1

MEAN

ST. DEV

359

16.2

368

14.6

359

11.3

360

8.0

DAY 8

WEEK 2

MEAN

ST. DEV

365

17.5

379

14.4

369

13.5

369

12.0

DAY 15

WEEK 3

MEAN

ST. DEV

379

20.3

394

17.5

380

15.0

382

12.2

FEMALES

GROUP 1

CONTROL

GROUP 2

3 MG/KG

GROUP 3

10 MG/KG

GROUP 4

30 MG/KG

PRE MATING

DAY 1

WEEK 1

MEAN

ST. DEV

216

5.5

221

10.6

222

6.6

221

10.2

DAY 8

WEEK 2

MEAN

ST. DEV

225

8.8

226

6.4

231

9.4

231

11.8

MATING PERIOD

DAY 1

WEEK 1

MEAN

ST. DEV

231

10.0

234

12.0

238

12.0

238

12.4

DAY 8

WEEK 2

MEAN

ST. DEV

254

---

250

---

DAY 15

WEEK 3

MEAN

ST. DEV

283

---

270

---

DAY 22

WEEK 4

MEAN

ST. DEV

333

---

322

---

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

FEMALES

F0-GENERATION

GROUP 1

CONTROL

GROUP 2

3 MG/KG

GROUP 3

10 MG/KG

GROUP 4

30 MG/KG

POST COITUM

DAY 0

MEAN

ST. DEV

229

11.0

233

11.4

239

12.5

240

10.2

DAY 4

MEAN

ST. DEV

245

8.6

251

14.0

254

10.0

251

9.9

DAY 7

MEAN

ST. DEV

253

10.9

257

17.0

260

12.6

263

12.69

DAY 11

MEAN

ST. DEV

266

10.1

272

16.6

273

15.9

278

13.0

DAY 14

MEAN

ST. DEV

277

11.5

284

19.7

286

14.7

290

13.6

DAY 17

MEAN

ST. DEV

301

12.1

309

22.5

308

13.3

317

16.1

DAY 20

MEAN

ST. DEV

335

17.1

347

27.0

343

19.5

355

25.5

LACTATION

DAY 1

MEAN

ST. DEV

262

14.8

262

20.1

274

14.0

271

16.3

DAY 4

MEAN

ST. DEV

266

13.0

274

19.3

284

17.2

272

14.2

DAY 7

MEAN

ST. DEV

280

12.7

283

20.8

289

18.6

283

15.6

DAY 13

MEAN

ST. DEV

297

11.2

294

24.4

302

17.3

299

19.6

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

MACROSCOPIC FINDINGS SUMMARY

MALES

GROUP 1

CONTROL

GROUP 2

3 MG/KG

GROUP 3

10 MG/KG

GROUP 4

30 MG/KG

END OF TREATMENT

Animals examined

Animals without findings

Animals affected

Stomach

Focus/foci

Irregular surface

Kidneys

Pelvic dilation

Epididymides

Nodule(s)

Preputial glands

Focus/foci

Thyroid gland

Discolouration

Agenesis

Thymus

Focus/foci

Mandibular lymph n

Discolouration

FEMALES

GROUP 1

CONTROL

GROUP 2

3 MG/KG

GROUP 3

10 MG/KG

GROUP 4

30 MG/KG

END OF TREATMENT

Animals examined

Animals without findings

Animals affected

Stomach

Focus/foci

Gelatinous

Kidneys

Cyst(s)

Discolouration

Clitoral glands

Focus/foci

Adrenal glands

Enlarged

Thymus

Focus/foci

Reduced in size

Gelatinous

Mandibular lymph n

Focus/foci

Parathymic lymph n

Discolouration

#/## Fisher’s Exact test significant at 5% (#) or 1% (##) level

Conclusions:

Executive summary:

Title

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of LOWINOX® 22IB46 in rats by oral gavage, including preliminary dose range finder

Guidelines

The study was based on the following guidelines:

• OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2015.

• OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

• OECD 421, Reproduction/Developmental Toxicity Screening Test, July 2015

• OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

• EC No 440/2008 B.7: "Repeated Dose (28 days) Toxicity (oral)", May 2008.

• OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.

• OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

Rationale for dose levels

Based on the results of a 10-day dose range finder, the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 3, 10 and 30 mg/kg.

Study outline

The test item, formulated in propylene glycol, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females.

Sampling and analysis of dose formulations was performed on two occasion during the treatment phase to show accuracy, homogeneity and stability of the test item in vehicle, propylene glycol.

Evaluated parameters

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment and PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues.

Results/discussion

Based on the results of formulation analysis it was considered that the formulations were prepared accurately and homogenously, and were stable for at least 5 hours at room temperature down to concentrations as low as 0.6 mg/mL (equivalent to a dose level of 3 mg/kg).

Parental results:

Uncoordinated movements were observed for a major part of the study period in both high dose males and females. In some of these females, temporary behavioural changes, including lethargy, restlessness or fearfulness, were occasionally observed. The functional observation test performed towards the end of the treatment period showed decreased fore limb grip strength in both high dose males and females and an accelerated decrease in movements and ambulations in the motor activity test in high dose females only in contrast to a more gradual decrease in activity in a normal habituation pattern. There might be a relation between the clinical signs and behavioural changes which might be interpreted as signs of neurotoxicity caused by treatment with LOWINOX® 22IB46 at 30 mg/kg/day. In the absence of changes in gait/motility and effects on motor activity in the mid dose animals, the minimal decreases in fore limb grip strength observed at this dose level were considered to be non-adverse and of no toxicological significance.

Small increases in liver weights were observed in high dose males. Histopathology of the liver revealed minimal hepatocellular hypertrophy, but in the absence of any degenerative findings this was considered to be a non-adverse finding.

Furthermore, histopathology also revealed follicular cell hypertrophy of the thyroid gland in high dose males. The minor increase in incidence and/or severity (up to slight degree) and the fact that it is usually an adaptive response to induction of hepatic enzymes, this finding was regarded to be an adaptive change and considered to be non-adverse.

No treatment-related and/or toxicologically significant changes were noted in any of the other parental parameters investigated in this study (i.e. body weight, food consumption, clinical laboratory investigations and macroscopic examination).

Conclusion

Treatment with LOWINOX® 22IB46 by oral gavage in male and female Wistar Han rats at dose levels of 3, 10 and 30 mg/kg revealed parental and developmental toxicity at 30 mg/kg/day. No reproduction toxicity was observed for treatment up to 30 mg/kg/day.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL: 10 mg/kg/day (based on clinical signs and behavioural changes observed in the functional observations tests).

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 10 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: K1
System:: other: affected moevment and grip strength
Organ:: other: not organ specific - affected movement and grip strength

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

Endpoint conclusion

Endpoint conclusion:: no study available

Additional information

Repeated dose toxicity: oral

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of LOWINOX® 22IB46 in rats by oral gavage, including preliminary dose range finder.

Rationale for dose levels

Study outline

Sampling and analysis of dose formulations was performed on two occasion during the treatment phase to show accuracy, homogeneity and stability of the test item in vehicle, propylene glycol.

Evaluated parameters

Results/discussion

Parental results:

Conclusion

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL: 10 mg/kg/day (based on clinical signs and behavioural changes observed in the functional observations tests).

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