Oxybis(methyl-2,1-ethanediyl) diacrylate

Endpoint summary

Description of key information

- Oral: Rat: combined 28-day repeated dose oral (gavage) toxicity study with the reproduction/developmental toxicity screening test (OECD TG 422): NOAEL (systemic) = 125 mg/kg bw/day

- Dermal: Rat: subchronic 3 months: NOAEL (systemic) = 66.66 mg/kg bw/day; LOAEL (local) = 20 mg/kg bw/day (Bio/Dynamics Inc. 1992, Val. 2); Read-across to CAS No. 42978-66-5

- Inhalation: No data available

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Mar - Aug 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliance

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:

July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed on August 2019

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: ReachCentrum SA
- Batch number of test material: 180003P040
- Expiration date of the lot/batch: 31. Dec 2018
- Purity/Composition: 100% (UVCB)
- Appearance: clear, colorless liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Stability under storage conditions: until 31. Dec 2018
- Stability of the test substance in the vehicle: Stability for at least 24 hours at room temperature protected from light, stability for at least 8 days in the refrigerator, and stability of 0.5 mL samples for at least 3 weeks in the freezer (≤ -15°C) is confirmed over the concentration range 1 to 200 mg/mL (solutions)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han), outbred, SPF-Quality

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. 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 ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks (males), 13 weeks (females)
- Weight at study initiation: males: 251- 322 g; females: 198- 263 g
- Housing: individually (females during the post-mating phase and lactation phase with the pups) and grouping (pretest, males during the post-amting phase
- Diet: ad libitum; pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum
- Acclimation period: for 5 days prior to start of the pretest period (females) or 5 days before the commencement of dosing (males).

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water is performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40-70 (daily mean relative humidity of 36 to 60%)
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
Test item dosing formulations (w/w) were homogenised to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 8, 25, 75 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed using a validated analytical procedure. Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.

Duration of treatment / exposure:

males: 29 days; females: 50-62 days
The duration of treatment covered a 2-week premating period and mating in both sexes as well as entire gestation and the duration of pregnancy and at least 14 days after delivery,
up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 40-53 days.

Frequency of treatment:

daily (7d/week)

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

125 mg/kg bw/day (nominal)

Dose / conc.:

375 mg/kg bw/day (nominal)

No. of animals per sex per dose:

- in total 10 animals/ sex/ dose
- 5 animals /sex/ dose were selected for functional tests (males only), clinical pathology, collection of full list of organs/tissues at macroscopic examination, organ weights (full list) and histopathology (full list)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 16-day dose range finder with oral gavage administration of the test item.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
F0 animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Pups were observed daily for general health/mortality. The number of live and dead pups were determined on PND 1 and daily thereafter.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily (F0 and pubs)

BODY WEIGHT: Yes
- Time schedule for examinations: F0: First day of treatment (prior to dosing) and weekly thereafter (after dosing); F1: Live pups were weighed individually on PND 1, 4, 7 and 13.
- Body Weight Gains: Calculated against the body weight on Day 1 (premating, mating and lactation periods) or Day 0 (postcoitum period).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Time schedule for examinations: quantitatively measured 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.
- Relative Food Consumption Calculated against the body weight for scheduled intervals.

WATER CONSUMPTION: not quantitative
- Subjective appraisal was maintained during the study

HAEMATOLOGY: Yes, F0 animals
- Time schedule for collection of blood: day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, males only (maximum of 24 hours)
- How many animals: 5/sex/group
- Parameters checked in table 1 were examined.

COAGULATION
Blood plasma of F0 animals was analyzed for Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, males only (maximum of 24 hours)
- How many animals: 5/sex/group
- Parameters checked in table 2 were examined.

OTHER:
Functional observational battery (FOB): Males only
- Functional tests were performed on the selected 5 males (F0) during Week 4 of treatment (after dosing, after completion of clinical observations)
- Examined parameters:
• Hearing ability
• Pubillary reflex
• Static righting reflex
• Fore- and hind-limb grip strength
• Locomotor activity

Estrous cycle:
- Daily vaginal lavage was performed for all females (F0) beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.

Female reproduction and delivery data
From the mating period onwards, the following parameters were recorded for each female (F0): male number paired with, mating date, confirmation of pregnancy and delivery day.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care

Thyroid hormones
- Time schedule for collection of blood: All F0 animals on scheduled necropsy, PND 14-16 and PND 4 for 2 pubs per litter
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: males only ( mximum of 24 h)
- How many animals: All F0 animals, 2 pubs per litter on PND 4 and PND 4-16
- For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no adverse changes in T4 were noted in F0- males, no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight were recorded
- Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16

OTHER:
Sex was externally determined for all pups on PND 1 and 4.
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
All male pups in each litter were examined for the number of areola/nipples on PND 13.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 3 and 4, Organ weights)

HISTOPATHOLOGY: Yes (see table 5 for collected tissue)
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
- Selected animals: Tissues identified in Text Table 5 (except animal identification, aorta, nasopharynx, esophagus, harderian
gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue).
- Males that failed to sire (except for males which were selected), females that failed to deliver pups and females with total litter loss: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.
- Females with total litter loss: Mammary gland.
- Remaining animals: Gross lesions/masses.

Statistics:

Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation seen after dosing among animals of all dose groups was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Further observed clinical signs affecting the skin/fur (alopecia, scabs), the eye (Chromodacryorrhoea) and breathing (rales) were observed.

Mortality:

no mortality observed

Description (incidence):

One female of the control group (no. 49) was euthanized on Lactation Day 1, as she had a total litter loss (at first litter check she had only dead pups).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight gain was increased in males in the second week of treatment after dosing of 125 and 375 mg/kg, and in the fifth week of treatment after dosing with 375 mg/kg. These changes in body weight gain were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment and values were within the historical control range

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significant change in relative food consumption in 40 mg/kg females during post-coitum Days 17-20 was considered to be unrelated to treatment, since no trend was apparent regarding dose and duration of treatment.

Food efficiency:

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A decreased number of reticulocytes was observed in females at 125 mg/kg, which was considered unrelated to administration of the test item due to absence of a dose-related trend response.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males: The following statistically significant increases were noted for treated males (relative changes in mean values as compared to the concurrent control group are indicated between parentheses):
-increased total protein at 375 mg/kg (6%)
-increased albumin at 375 mg/kg (6%)
-increased calcium at 375 mg/kg (4%)
-increased urea at 40 and 375 mg/kg (27% and 30%, respectively)
The changes in total protein, albumin and calcium were minimal and remained within the historical control range. No dose related trend was observed for the increase in urea and values remained within the historical control range. In addition, a slight increase in sodium was observed at 125 mg/kg (1%), which occurred in the absence of a dose related trend.

No treatment-related changes were noted in clinical chemistry parameters in females

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item-related higher liver weights (absolute and relative to body weights) were noted in the 375 mg/kg/day group males.
There were no other test item-related organ weight changes.
The significant relative prostate gland weight decrease and liver weight increase in the 40 mg/kg/day treated males was considered incidental and not related to treatment in absence of a dose-related trend.

for details see table 6

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related irregular surface was observed in the (fore)stomach in 2/10 males treated at 125 mg/kg/day and in 10/10 males and 2/10 females treated at 375 mg/kg/day.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Watery fluid in the uterus, found in one control female and 3 females treated with 125 mg/kg, is related to a stage in the estrous cycle and is a normal finding.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the (fore)stomach of males and females and the liver and kidneys of males.

Stomach, squamous cell hyperplasia was present in 2/5 males starting at 125 mg/kg/day up to marked degree and in females at 375 mg/kg/day up to moderate degree. This correlated with the macroscopic irregular surface.
Stomach, ulcer forestomach was present in males starting at 125 mg/kg/day at minimal degree.
Stomach, inflammation forestomach was present in males starting at 125 mg/kg/day up to moderate degree.

Liver, hepatocellular hypertrophy was present in males treated at 375 mg/kg/day at minimal degree. This correlated with the increased liver weight. Due to the absence of any indicators of cellular degeneration. The changes in the liver were not considered adverse at current severities.

Kidneys, an increased incidence and severity of hyaline droplet accumulation was present in males treated at 375 mg/kg/day up to slight degree. The increased hyaline droplet accumulation in the male kidneys was not accompanied by indicators of tubular damage and therefore this was considered to be nonadverse.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

- Functional observation parameters were not considered to be affected by treatment in males up to 375 mg/kg.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Forelimb and hind limb grip strength was similar between control and treated animals. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
- Coagulation parameters (prothrombin time and activated partial thromboplastin time) of treated rats were considered not to have been affected by treatment.
-Serum levels of T4 in F0 males were increased at 125 and 375 mg/kg (35% and 37% increase in mean values compared to concurrent control, respectively). These values remained within the historical control range and the control value was on the lower limit of this range. In addition, no effect was observed in respect to thyroid weight, therefore, this effect was considered not toxicologically relevant.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

375 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects up to and including the highest tested dose

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

no

The various analyses confirmed

- Accuracy: The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulation.

- Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Table 6: Mean Percent Liver Weight Differences from Control Groups

Dose level in mg/kg bw/d	Males			Females
	40	125	375	40	125	375
LIVER
absolute	13	4	31**	-7	-4	1
Relative to bodyweight	9*	5	25**	-6	-1	0

*: P < 0.05, **: P < 0.01

Table 7: Summary Test Item-Realted Microcopic Findings-(fore)stomach

Dose level in mg/kg bw/d	Males				Females
	0	40	125	375	0	40	125	375
STOMACH^a	5	5	5	10	5	5	5	5
Hyperplasia squamous cell
Slight	-	-	-	-	-	-	-	1
Moderate	-	-	2	6	-	-	-	1
Marked	-	-	-	4	-	-	-	-
Ulcer forestomach
minimal	-	-	1	2	-	-	-	-
Inflammation forestomach
Minimal	-	-	1	6	-	-	-	-
Moderate	-	-	1	-	-	-	-	-

a = Number of tissues examined from each group

Table 8 Summary Test Item-Related Microscopic Findings – Liver and Kidneys – Males

	Males
Dose level in mg/kg bw/d	0	40	125	375
LIVER^a	5	5	5	5
Hepatocellular hypertrophy
minimal	-	-	-	4
KIDNEY^a	5	5	5	5
Hyaline droplet accumulation
minimal	1	1	2	3
slight	-	-	-	2

a= Number of tissues examined from each group

Conclusions:

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) were established:
Parental local NOAEL: 40 mg/kg (based on findings in the (fore)stomach)
Parental systemic NOAEL: at least 375 mg/kg due to the absence of adverse toxicity in the study for both sexes.

Executive summary:

Wistar Han rats were treated with the test item by daily oral gavage at dose levels of 40, 125 and 375 mg/kg according to OECD 422 and in compliance with GLP. The rats of the control group received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-62 days). Females that failed to deliver pups were treated for 40-53 days.

Parental toxicity was observed in the (fore)stomach of males from 125 mg/kg and in females at 375 mg/kg.

These changes consisted of ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females.

Other treatment-related but non-adverse changes were observed in the liver at microscopic examination. An absolute increase of 31% and a relative increase of 25% in liver weight was observed at dose 375 mg/kg. At microscopic examination, hepatocellular hypertrophy in the liver was observed at minimal severity and was in the absence of any indicators of cellular degeneration. The changes in the liver were not considered adverse at current severities. In the kidneys an increase in hyaline droplet accumulation was recorded in males which was considered to likely represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules (Alden et al., 1991). This male rat specific protein is not present in female rats nor in higher mammals, including man (Sahota et al., 2013). The increased hyaline droplet accumulation in the male kidneys at 375 mg/kg/day was not accompanied by indicators of tubular damage and therefore this was considered to be nonadverse.

Functional observations were not performed for females and therefore, possible treatment related effects on the functional parameters could not be evaluated.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations (males), body weight, food consumption, clinical laboratory investigations (including male T4 thyroid hormone levels), macroscopic examination and organ weights).

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, no systemic no-observed-adverse-effect level (NOAEL) were established under the conditions of this study. The Parental local NOAEL of 40 mg/kg is based on the findings in the (fore)stomach. Due to the absence of systemic toxicity up to and including the highest tested dose of 375 mg/kg bw/d no systemic NOAEL could be derived.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07/2020 to 04/2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Jul 2016

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

Jul 2000

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 421 (Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

Jul 2016

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

Jul 2000

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

May 2008

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

Oct 2008

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3050 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

Jul 2000

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

CAS no: 57472-68-1

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle, Cedex, France.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 - 12 weeks old for males and 12 - 14 weeks old for females.
- Weight at study initiation: 250 - 350 g for males and 200 - 250 g for females
- Housing: On arrival and following the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Makrolon plastic cages (MIII type, height 18 cm). During the lactation phase, females were housed in Makrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (dimensions: 48.3 x 26.7 x 20.3 cm) without cage enrichment, bedding material, food and water. Animals were separated during designated procedures/activities.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY
- The feed was analysed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water was performed, and results of these analyses are on file at the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Aug 2020 To: 13 Nov 2020

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Rate of preparation of dosing solutions: weekly
- Preparation of dosing solutions: Test item dosing formulations (w/w) were homogenised to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator, stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item. Any residual volumes were discarded.
- Storage temperature of dosing solutions: Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.

VEHICLE
- Concentration in vehicle: 8, 25, 75 mg/mL
- Amount of vehicle: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Samples for Analysis: Duplicate middle samples for Groups 1 and 3 (concentration analysis only) and duplicate top, middle, and bottom samples for Groups 2 and 4 (concentration and homogeneity analysis).
- Sample Volume: Approximately 500 mg accurately weighed.
- Acceptance Criteria: For concentration, the criteria for acceptability were mean sample concentration results within or equal to ± 10 % for solutions or ± 15 % for suspensions of target concentration. For homogeneity, the criteria for acceptability were a coefficient of variation (CV) of concentrations of ≤ 10 % for each group.

Duration of treatment / exposure:

Males for 29 days, females that delivered for 50 - 62 days, females which failed to deliver or had a total litter loss for 39 - 44 days.

Frequency of treatment:

Once a day, seven days a week.

Dose / conc.:

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

Remarks:

Group 2. Low dose.

Dose / conc.:

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

Remarks:

Group 3. Mid dose.

Dose / conc.:

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

Remarks:

Group 4. High dose.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 10-day dose range finder with oral gavage administration of DPGDA in rats (Test Facility Study No. 20236861) and in an attempt to produce graded responses to the test item. The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
- Fasting period before blood sampling for clinical biochemistry: Males (except for animals which were sacrificed in extremis or found dead) were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. Females were not fasted.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During treatment, animals were observed at least once daily, up to the day prior to necropsy.

DETAILED CLINICAL OBSERVATIONS: Yes
- Animals were observed for specific clinical signs. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade were predefined at 1, 2, 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 (i.e. maximum grade 1) were scored.
Once before the first administration of the test item and at weekly intervals during the treatment period, animals were observed for specific clinical signs in a standard arena. The time of onset, grade and duration of any observed signs were recorded.

CLINICAL OBSERVATIONS (FUNCTIONAL OBSERVATION BATTERY TESTS)
- Time schedule: Once during the treatment period. The selected 5 males were tested once during Week 4 of treatment and the selected 5 females were tested once during the last week of lactation (i.e. Postnatal day (PND) 6 - 13). These tests were performed after clinical observations (including arena observation, if applicable).
The following tests were performed:
(1) Hearing ability, pupillary reflex and static righting reflex (score 0 = normal/present, score 1 = abnormal/absent).
(2) Fore- and hind-limb grip strength were recorded as the mean of three measurements, using a grip strength meter.
(3) Locomotor activity (recording period: 1 hour under normal laboratory light conditions) were tested using the Kinder Scientific Motor Monitor System. Total movements and ambulations were 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.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to 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. In order to monitor the health status animals may be weighed more often.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
Weekly, except for males and females which are housed together for mating and for females without evidence of mating. Food consumption of mated females were measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. Food consumption were quantitatively measured.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Regular basis throughout the study.
Water consumption were monitored by visual inspection of the water bottles. If inter group differences are noted, consumption may be assessed by weight.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood were collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus in the animal facility. Additional blood samples may be obtained (e.g. due to clotting of non-serum samples) in both the animal facility and in the necropsy room if permissible sampling frequency and blood volume are not exceeded. After collection, samples were transferred to the appropriate laboratory for processing.
- Anaesthetic used for blood collection: Yes, isoflurane.
- Animals fasted: Males (except for animals which were sacrificed in extremis or found dead) were fasted overnight with a maximum of 24 hours before blood sampling, but water were available. Females will not be fasted overnight.
- How many animals: All parental animals (except for animals which were sacrificed in extremis or found dead and females with total litter loss.
- Parameters examined: white blood cell count (WBC), reticulocytes (absolute), neutrophils (absolute), red blood cell distribution width (RDW), lymphocytes (absolute), haemoglobin, monocytes (absolute), haematocrit, eosinophils (absolute), mean corpuscular volume (mcv), basophils (absolute), mean corpuscular haemoglobin (MCH), large unstained cells (LUC) (absolute), red blood, cell count mean corpuscular, haemoglobin concentration (MCHC) and platelets.
For Haematology: Target volume: 0.5 mL; anticoagulant used: K3-EDTA (tubes; Greiner Bio-One GmbH, Kremsmünster, Austria). A blood smear were prepared from each haematology sample. Blood smears were labelled, stained, and stored. Blood smears were evaluated when required to confirm analyser results.
For Coagulation: Target Volume: 0.45 mL and anticoagulant used: Citrate (tubes; Greiner Bio-One GmbH, Kremsmünster, Austria). Coagulation Parameters: Prothrombin time (PT and Activated partial Thromboplastin Time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see Haematology.
- Animals fasted: see Haematology.
- How many animals: see Haematology.
- Parameters examined: Alanine aminotransferase (ALT) Creatinine, Aspartate aminotransferase (AST) Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total Bilirubin Chloride, Bile Acids, Calcium, Urea and Inorganic Phosphate (Inorg. Phos).
For Clinical Chemistry: Target volume: 0.5 mL; anticoagulant: Li-Heparin (tubes; Greiner Bio-One GmbH, Kremsmünster, Austria). Processing: to plasma

SERUM HORMONES: Yes
- Time of blood sample collection: see Haematology.
- Animals fasted: see Haematology.
- How many animals: see Haematology. Measurement of total T4 was conducted for parental males.
- Parameters examined: Thyroid Hormone Parameters Thyroxine (T4) and Thyroid-Stimulating Hormone (TSH; only if required)
For Thyroid Hormones: Target Volume: 1.0 mL; anticoagulant: not applicable for serum (tubes; Greiner Bio-One GmbH, Kremsmünster, Austria). After clotting and centrifugation, serum has been used.
For males, 150 μL serum for measurement of total T4 and the remaining. For females, the serum will be used for possible future measurement of total T4 and/or thyroid-stimulating hormone TSH.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
EUTHANASIA
Scheduled deaths: animals surviving until scheduled euthanasia were weighed, and deeply anesthetised using isoflurane and subsequently exsanguinated and subjected to a full post-mortem examination.
Unscheduled deaths: if necessary for humane reasons, animals were euthanised as per Test Facility SOPs. These animals were deeply anesthetised using isoflurane and subsequently exsanguinated. They underwent necropsy, and specified tissues were retained but not weighed.

NECROPSY
Scheduled necropsies are summarised below:
- Males (which sired or failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
- Females which delivered: PND 14 - 16.
- Females which failed to deliver: With evidence of mating: Post-coitum Days 26 - 27
- Females with total litter loss: Dams with no surviving pups were euthanised within 24 hours after the last pup is found dead or missing.
All animals were subjected to a full post-mortem examination, with special attention being paid to the reproductive organs. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

ORGAN WEIGHTS ANIMALS
The organs identified below were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals euthanised in poor condition or in extremis. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.
- Organs weighed at necropsy for all selected animals: cervix*; epididymis#; gland adrenal#; gland coagulation#$; gland parathyroid@; gland prostate; gland seminal vesicle#; gland thyroid#; heart; kidney#; liver; ovaries#; spleen; testes#; thymus and uterus, where:
* = weighed together with the uterus; # = paired organ weight; $ = weighed together with the seminal vesicles; @ = weighed together with the thyroid.
- Organs weighed at necropsy for all remaining animals (incl. Males that Failed to Sire, Females that Failed to Deliver and Females with Total Litter Loss) were as follows: epididymis*; gland coagulation*#; gland parathyroid$; gland prostate; gland seminal vesicle*; gland thyroid*; testes*, where:
* = Paired organ weight; # = Weighed together with the seminal vesicles; $ = Weighed together with the thyroid.

TISSUES FIXED AND PRESERVED
Representative samples of the tissues identified in the table below were collected from all animals and preserved in 10 % neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated.
- Tissue Collection and Preservation for all Selected Animals, all Animals that were Sacrificed in extremis and Females with Total Litter Loss: Animal identification: Artery; aorta; body cavity; nasopharynx; bone marrow; bone, femur; bone, sternum; brain (eight levels); cervix; epididymides*; oesophagus; eye*; gland, adrenal; gland, coagulation; gland, harderian* #; gland, lacrimal; gland, mammary; gland, parathyroid$; gland, pituitary; gland, prostate; gland, salivary; gland, seminal vesicle; gland, thyroid; gross lesions/masses; gut-associated lymphoid tissue; heart; kidney; large intestine, cecum; large intestine, colon; large intestine, rectum; larynx; liver; lung; lymph node (mandibular and mesenteric site); muscle, skeletal; nerve, optic#; nerve, sciatic; ovaries; pancreas; skin; small intestine, duodenum; small intestine, ileum; small intestine, jejunum; spinal cord; spleen; stomach; testes*; thymus; tongue; trachea; urinary bladder; uterus and vagina; where:
* = Preserved in modified Davidson’s fixative and transferred to formalin after fixation for at least 24 hours; # = Only collected if present in the routine section of the eye. Part of the optic nerve attached to the eye was Fixed in Modified Davidsons’s fixative. The remaining part of the optic nerve was placed in formalin; $ = Only collected if present in the routine section of the thyroid.
- Tissue Collection and Preservation for all Remaining Animals (incl. Males that Failed to Sire and females that failed to deliver): animal identification
cervix; epididymis*; gland, coagulation; gland, mammary; gland, parathyroid#; gland, pituitary; gland, prostate; gland, seminal vesicle; gland, thyroid; gross lesions/masses; ovaries; testes*; uterus and vagina, where:
* = Preserved in modified Davidson’s fixative and transferred to formalin after fixation for at least 24 hours; # = Only collected if present in the routine section of the thyroid.

HISTOPATHOLOGICAL EXAMINATION
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with haematoxylin and eosin:
- Selected animals, and unscheduled deaths (sacrificed in extremis): see Tissues identified in above for “Tissue Collection and Preservation for all Selected Animals, all Animals that were Sacrificed in extremis and Females with Total Litter Loss” (except animal identification, aorta; nasopharynx; oesophagus; harderian gland; lacrimal gland; salivary gland; larynx; optic nerve; pancreas; skin and tongue).
- Males that failed to sire, females that failed to deliver pups and females with total litter loss: cervix; epididymis; coagulation gland; prostate gland; seminal vesicles; ovaries; testes; uterus and vagina.
- Females with total litter loss: Mammary gland.
- Remaining animals: Gross lesions/masses.

Statistics:

All statistical tests were conducted at the 5 % significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1 % or 5 % levels. Numerical data collected on scheduled occasions for the listed variables were analysed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or % CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
- Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.
-Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
- Incidence: An overall Fisher’s exact test was used to compare all groups at the 5 % significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Two females at 375 mg/kg/day, showed piloerection on respectively Days 8 - 9 post-coitum and Days 3 - 4 of lactation. Another female also had a slightly pale and/or lean appearance between Days 2 and 5 of lactation, accompanied by a body weight loss up to 12 %. As clinical signs were transient and occurred in one or two animals only, these were considered incidental and not toxicologically relevant.
Salivation seen after dosing among males and/or females of all treatment groups was considered to be a local test item effect and likely resulting from irritating properties of the test item. Any other clinical signs noted during the treatment period (i.e. alopecia, scabs, rales and swelling of the vagina) 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 and/or did not show any apparent dose-related trend. At the incidence observed, these were considered not to be signs of toxicological relevance. No findings were noted during the weekly arena observations in this study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight gain in males at 375 mg/kg/day was decreased compared with control on Day 8 of treatment, which recovered up to similar levels as control at the end of the mating period. Absolute body weights in males at 375 mg/kg/day were lower compared with control from start of treatment onwards (0.97x of control on Day 1 of treatment down to 0.95x of control on Day 15 of the mating period). Body weights and body weight gain in females were considered to have been unaffected by treatment with the test item. Considering the magnitude of change and as Group 4 males just happened to be the smallest rats in this study, no toxicological relevance was attached to this finding.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption before or after correction for body weight was considered unaffected by treatment with the test item up to 375 mg/kg/day. A trend towards lower food consumption was recorded in males and females at 375 mg/kg/day during the first week of treatment, with subsequent complete recovery. No toxicological relevance was attached to this finding.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At 375 mg/kg/day, the following changes in haematology parameters were recorded (statistically significant unless stated otherwise): (1) Increased neutrophil counts in males and females (1.83x and 1.20x of control, respectively; statistically significant in males only). This was considered test item-related. (2) Decreased mean corpuscular haemoglobin concentration (MCHC) in females (0.95x of control). In the absence of changes in correlating parameters, this was considered not related to treatment with the test item. The decreased platelet counts in males at 40 mg/kg/day were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend. Coagulation parameters of treated rats were considered not to have been affected by treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following changes in clinical biochemistry parameters were recorded in females at 375 mg/kg/day: (1) Increased cholesterol levels in females at 375 mg/kg/day (1.38x of control). This was considered test item-related. (2) Increased calcium levels in females at 375 mg/kg/day (1.07x of control). This was considered test item-related. The increased mean glucose concentration recorded in females at 40 mg/kg/day was considered to be unrelated to treatment with the test item as it occurred in the absence of a dose-related trend. No toxicologically relevant changes were noted in clinical biochemistry parameters in males.

Endocrine findings:

no effects observed

Description (incidence and severity):

Thyroid hormone analyses: Serum levels of T4 in parental males were considered unaffected by treatment with the test item up to 375 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals up to 375 mg/kg/day. Motor activity was considered to be unaffected by treatment with the test item. The higher mean values of total movements and ambulations in males at 125 mg/kg/day (not statistically significant) were caused by two animals only. Increased motor activity in females at 125 mg/kg/day was mainly apparent in intervals 5 - 7, after which the habituation profile recovered to being comparable with controls. In the absence of a dose-related trend, this was considered not related to treatment with the test item. All groups (except Group 3 females) showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weights and organ: body weight ratios were considered unaffected by treatment with the test item. Lower adrenal gland weights (absolute and relative to body weight) were recorded for females at 375 mg/kg/day. In the absence of a histopathological correlate, this weight change was regarded unrelated to the treatment with the test item. In males at 375 mg/kg/day, some organ weight differences reached statistical significance when compared to the control group: lower absolute spleen weight, higher relative weights for brain, heart and testes. These weight changes were considered to be the result of a test item-related effect on final body weight (0.93x of control). Remaining changes which reached statistical significance (lower absolute weight of prostate gland at 125 mg/kg/day; lower absolute epididymis weight at 40 mg/kg/day) were considered not to be test item-related due to the lack of a dose-related pattern and/or general overlap and variability in individual values.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related irregular surface of the forestomach was recorded in 1/10 males at 125 mg/kg/day and in 9/10 males and 7/10 females at 375 mg/kg/day. The microscopic correlate for this finding was squamous cell hyperplasia and hyperkeratosis of the forestomach. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with DPGDA were noted in the stomach of both sexes and liver of females. A combination of findings was recorded for the forestomach and consisted of mixed cell inflammation (up to slight), squamous cell hyperplasia (up to marked) and/or hyperkeratosis (up to moderate) in most animals at 375 mg/kg/day. Low degrees of squamous cell hyperplasia and hyperkeratosis was recorded for the forestomach in a single male at 125 mg/kg/day. In addition, a forestomach ulcer at minimal degree was recorded in a single male at 125 and at 375 mg/kg/day and in a single female at 375 mg/kg/day.
In two females at 375 mg/kg/day, a minimal degree of increased mitotic figures was recorded for the liver. 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

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

haematology

histopathology: non-neoplastic

Key result

Critical effects observed:

no

VERIFICATION OF TEST SOLUTIONS

Accuracy: During analysis of Week 3 formulations the extraction of the test item with methanol was most likely insufficient, therefore formulations analysis was repeated with Week 4 formulations including a more vigorous extraction. The analysis of Week 4 formulations was considered representative. The concentrations analysed in the Week 4 formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90 % and 110 %). A small response at the retention time of the test item was observed in the chromatograms of the Group 1 formulations prepared for use in Weeks 3 and 4. The maximum contribution to the Group 2 samples was 0.14 % and 0.076 %, respectively.

Homogeneity: the formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10 %).

Hence, formulation analyses showed that formulations of test item in corn oil were prepared accurately and homogenously.

Table 1Summary of Haematology Values: F0 Generation -Day: 30 Relative to Start Date (males)

Sex: Male		Reporting Haematology
		WBC (10^9/L) [G]	NEUT (10^9/L) [G]	LYMPH (10^9/L) [G]	MONO (10^9/L)  [G1]	EOS (10^9/L) [G1]	BASO (10^9/L) [G]	LUC (10^9/L) [G]	RBC (10^12/L) [G]	RETIC (10^9/L) [G]	RDW (%) [G]	H B (g/L) [G]	HCT (L/L) [G]	MCV (fL)	MCH (pg) [G2]	MCHC (g/L) [G2]	PLT  (10^9/L) [G2]
0 mg/kg/day  Group 1	Mean	7.088	0.900	5.962	0.100	0.084	0.012	0.028	8.786	228.18	12.90	156.4	0.4590	52.24	17.80	340.6	736.0
	SD	1.774	0.153	1.697	0.037	0.021	0.004	0.016	0.264	30.20	0.89	5.1	0.0129	1.18	0.52	4.0	40.3
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
40 mg/kg/day Group 2	Mean	6.080	1.014	4.886	0.090	0.058	0.006	0.024	8.816	262.34	13.04	156.4	0.4654	52.78	17.74	336.2	638.6 *
	SD	1.075	0.252	0.869	0.034	0.019	0.005	0.009	0.270	45.21	0.49	4.2	0.0134	1.18	0.49	4.9	58.8
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
	tCtrl	0.86	1.13	0.82	0.90	0.69	0.50	0.86	1.00	1.15	1.01	1.00	1.01	1.01	1.00	0.99	0.87
125 mg/kg/day  Group 3	Mean	5.660	0.828	4.634	0.104	0.058	0.008	0.034	8.840	217.94	12.36	156.4	0.4618	52.24	17.70	338.4	686.8
	SD	0.979	0.191	0.877	0.027	0.024	0.008	0.017	0.309	36.31	0.59	3.5	0.0129	1.23	0.56	4.6	36.1
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
	tCtrl	0.80	0.92	0.78	1.04	0.69	0.67	1.21	1.01	0.96	0.96	1.00	1.01	1.00	0.99	0.99	0.93
375 mg/kg/day  Group 4	Mean	7.840	1.644**	5.886	0.140	0.110	0.018	0.044	8.742	273.04	12.98	153.4	0.4602	52.68	17.54	333.4	734.0
	SD	2.649	0.489	2.229	0.073	0.101	0.008	0.023	0.293	34.88	0.79	3.8	0.0151	0.64	0.39	5.2	75.2
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5

 [G] - Anova & Dunnett: ** = p ≤ 0.01

[G1] - Kruskal-Wallis & Dunn

[G2] - Anova & Dunnett: * = p ≤ 0.05

Table 2 Summary of Haematology Values: F0 Generation - Day: 51 Relative to Start Date (females)

Sex: Female		Reporting Haematology
		WBC (10^9/L) [G]	NEUT (10^9/L) [G]	LYMPH (10^9/L) [G]	MONO(10^9/L) [G]	EOS 10^9/L) [G1]	BASO 10^9/L) [G]	LUC 10^9/L) [G]	RBC 10^12/L) [G]	RETIC 10^9/L) [G]	RDW (%) [G]	H B(g/L) [G]	HCT L/L) [G]	MCV (fL) [G]	MCH (pg) [G2]	MCHC (g/L) [G2])	PLT (10^9/L) [G2]
0 mg/kg/day  Group 1	Mean	5.462	1.706	3.542	0.098	0.090	0.012	0.012	7.064	226.84	13.12	139.2	0.4076	57.78	19.76	341.8	690.6
	SD	0.657	0.371	0.428	0.024	0.063	0.004	0.004	0.363	53.05	1.51	3.8	0.0127	3.50	1.03	9.0	69.8
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
40 mg/kg/day Group 2	Mean	5.288	1.652	3.412	0.098	0.096	0.010	0.018	7.166	237.84	12.46	135.8	0.4042	56.46	19.02	337.0	690.8
	SD	0.906	0.452	0.540	0.016	0.054	0.000	0.004	0.423	45.53	0.50	3.5	0.0154	1.85	0.85	9.6	65.9
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
	tCtrl	0.97	0.97	0.96	1.00	1.07	0.83	1.50	1.01	1.05	0.95	0.98	0.99	0.98	0.96	0.99	1.00
125 mg/kg/day  Group 3	Mean	5.136	1.602	3.348	0.106	0.062	0.006	0.012	6.950	252.76	13.04	136.4	0.4056	58.44	19.64	335.8	683.0
	SD	0.620	0.226	0.636	0.027	0.008	0.005	0.004	0.287	30.47	0.91	5.2	0.0156	2.86	1.01	3.6	60.3
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
	tCtrl	0.94	0.94	0.95	1.08	0.69	0.50	1.00	0.98	1.11	0.99	0.98	1.00	1.01	0.99	0.98	0.99
375 mg/kg/day  Group 4	Mean	6.188	2.046	3.880	0.132	0.090	0.010	0.030	6.650	283.98	14.44	133.4	0.4088	61.86	20.18	326.4 **	650.8
	SD	2.649	0.489	2.229	0.073	0.101	0.008	0.023	0.293	34.88	0.79	3.8	0.0151	0.64	0.39	5.2	75.2
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
	tCtrl	1.11	1.83	0.99	1.40	1.31	1.50	1.57	0.99	1.20	1.01	0.98	1.00	1.01	0.99	0.98	1.00

[G] - Anova & Dunnett

[G1] - Kruskal-Wallis & Dunn

[G2] - Anova & Dunnett: ** = p ≤ 0.01

Table 3 Macroscopic findings summary

MALES	Group 1 Control	Group 2 40 mg/kg/day	Group 3 125 mg/kg/day	Group 4 375 mg/kg/day
END OF TREATMENT Animals examined	10	10	10	10
Animals without findings	9	10	8	1
Animals affected	1	0	2	9
Stomach - Irregular surface	0	0	1	9 ##
Liver- Accentuated lobular pattern	0	0	0	1
Thyroid - gland Enlarged	0	0	1	0
Thyroid - Discolouration	1	0	0	0
FEMALES	Group 1 Control	Group 2 40 mg/kg/day	Group 3 125 mg/kg/day	Group 4 375 mg/kg/day
INTERCURRENT DEATH Animals examined	1	1		1
Animals affected	1	1		1
General observations - Incomplete delivery.	0	1		0
Total litter loss	1	0		1
Stomach Irregular surface	0	0		1
Stomach Discolouration	0	0		1
Stomach Gelatinous	0	0		1
Stomach Discolouration	0	1		0
Liver Discolouration	0	0
Uterus Outside uterus 3 pups dead 2 males, no abnor.	0	1		0
Thyroid gland Discolouration	0	1		0
END OF TREATMENT Animals examined	9	9	10	9
Animals without findings	5	6	8	3
Animals affected	4	3	2	6
Stomach Irregular surface	0	0	0	6 ##
Uterus Contains fluid	1	0	0	0
Clitoral glands Focus/foci	1	0	0	0
Discolouration	0	0	1	0
Thymus Focus/foci	0	1	0	0
Mesenteric lymph n Enlarged	0	0	0	1
Discolouration	0	0	0	1
Mandibular lymph n
Focus/foci	0	1	0	0
Enlarged	0	1	0	0
Skin Alopecia	1	1	1	0
Eyes Exophthalmus	1	1	0	0

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

Conclusions:

In a combined 28-Day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD TG 422), based on the occurrence of macroscopic and microscopic stomach findings and increased neutrophil counts of the high dose animals, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 125 mg/kg bw/day for males and females.

Executive summary:

The objective of this OECD TG 422 study performed in compliance with GLP was to determine the potential toxic effects of DPGDA. Wistar Han rats were administered the test substance dose levels of 0, 40, 125, 375 mg/kg/day, based on the results of the Dose Range Finder. With the exception of the control group, all animals (10 animals/sex/group) received the test substance orally via gavage for a minimum period of 28 days using corn oil as vehicle at a dose volume of 5 mL/kg body weight. Chemical analyses of formulations were conducted twice during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, clinical pathology, measurement of thyroid hormone T4 (parental males), gross necropsy findings, organ weights and histopathologic examinations.

Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously. Parental toxicity was observed at the highest dose level tested (375 mg/kg/day). One female at 40 mg/kg/day was sacrificed in extremis on post-coitum Day 23 because of delivery difficulties. This female delivered 3 dead pups and showed hunched posture, piloerection and pale appearance. At necropsy, 8 dead foetuses were present in the uterus. Microscopically the uterus showed characteristics of pregnancy. Findings regarded secondary to the presence of dead foetuses in the uterus were moderate multifocal hepatocellular necrosis of the centrilobular area of the liver and multifocal thrombi in the lungs. These findings in liver and lungs were attributed to the poor condition of this female. This death was regarded to be related to delivery difficulties and unrelated to the treatment with the test item.

Significant body weight loss in one female at 375 mg/kg/day during the first days of lactation coincided with piloerection and a slightly pale and lean appearance. At the incidence observed, this was considered not toxicologically relevant. Body weight gain in males at 375 mg/kg/day was decreased compared with control on Day 8 of treatment, which recovered up to similar levels as control at the end of the mating period. Absolute body weights in males at 375 mg/kg/day were decreased compared with control from Day 1 of treatment up to the day of scheduled necropsy. Considering the magnitude of change and as Group 4 males just happened to be the smallest rats in this study, no toxicological relevance was attached to this finding.

Relationships were suspected between gross necropsy (irregular surface of the forestomach), clinical pathology (increased neutrophil concentrations) and histopathology observations. The combination of microscopic stomach findings observed in most males and females treated at a dose of 375 mg/kg/day consisted of slight to marked squamous cell hyperplasia with slight to moderate hyperkeratosis and minimal to slight mixed cell inflammation. In addition, a forestomach ulcer at minimal degree was recorded in a single male at 125 and at 375 mg/kg/day and in a single female at 375 mg/kg/day. This combination of findings is considered adverse at the recorded incidences and severities. Mixed cell inflammation was recorded in all selected males, compared to 3/5 selected females and likely correlated to the increased neutrophils recorded for males at 375 mg/kg/day. The forestomach findings and salivation were considered to be local test item effects and likely resulting from irritating properties of the test item. The low severities of stomach findings in a single male at 125 mg/kg/day only were considered to be non-adverse. Increased mitotic figures were recorded in the liver of two females at 375 mg/kg/day and a relation to treatment with the test item could not be excluded. This alteration was considered to be non-adverse at the recorded minimal severity.

Non-adverse changes were recorded in clinical biochemistry parameters: increased cholesterol levels and increased calcium levels in females at 375 mg/kg/day. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, body weight, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), food consumption, clotting parameters, male T4 thyroid hormone levels, and organ weights). No reproductive toxicity was observed up to 375 mg/kg/day.

In conclusion, based on the results of this combined 28-Day repeated dose toxicity study the established No Observed Adverse Effect Level (NOAEL) of DPGDA (CAS 57472-68-1) was 125 mg/kg/day based on macroscopic and microscopic stomach findings and increased neutrophil counts).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

125 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant OECD TG 422 study

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jun - Sep 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 424 (Neurotoxicity Study in Rodents)

Principles of method if other than guideline:

The subchronic dermal application of the test substance to the backs of Sprague-Dawley rats was studied to assess potential neurotoxic and other local and systemic effects.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): C-178
- Physical state: liquid
- Analytical purity: 100% active ingredient
- Storage condition of test material: room temperature

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 41 days (age: 28 days at receipt)
- Weight at study initiation: week 0 males: 157-163 g; week 0 females: 133-139 g
- Housing: individually in elevated stainless steel cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

other: not occluded

Vehicle:

corn oil

Details on exposure:

TEST SITE
- Area of exposure: the back of the rats
- % coverage: no data
- Type of wrap if used: not occluded no further data
- Time intervals for shavings or clipplings: all animals were clipped ca. 23 h prior to initial dose. The animals were reclipped when necessary.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.077 ml/kg
- Concentration (if solution): 1.0, 3.33 and 10.0 %
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): immiscible with water
- Amount(s) applied (volume or weight with unit): 2.077 ml 7kg of the test substance in corn oil
- Concentration (if solution): 1.0, 3.33 and 10.0 % of the test substance in corn oil

USE OF RESTRAINERS FOR PREVENTING INGESTION: no data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

the remaining samples of weekly dosing solutions for each dose were returned to the sponsor for analysis, no further data

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily, 5 days/week

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

66.666 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: for mortality and gross signs of toxicologic or pharmacologic effects

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly including signs of local or systemic toxicity, pharmacologic effects and palpation for tissue masses

BODY WEIGHT: Yes
- Time schedule for examinations: twice pretest, weekly during treatment and terminally (after fasting)


OTHER:
- blood was obtained from over night fasted rats via venipuncture of the orbital sinus under light ether anestehsia, the same animals were used that were intended for formalin-fixation (5/sex/dose).
- hematology upon termination: hemoglobin, hematocrit, erythrocytes, clotting time, total and differential, leukocytes, erythrocytes morphology
- clinical chemistry upon termination: serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, blood urea nitrogen, fasting glucose, total protein, total bilirubin, sodium, potassium, calcium, inorganic phosphorus
- urinanalysis 6 days before termination: gross appearance, specific gravity, pH, protein, glucose, occult blood

Sacrifice and pathology:

One-half of the animals were sacrificed by exsanguination under light ether anesthesia, and selected organs and tissues were fixed
in formalin. Organ and organ-body weight ratios (adrenals, brain, liver, kidney, heart, spleen, testes with epididymides, ovaries) were determined on all of these animals only. Histopathological evaluations of 10 organs or tissues (liver, kidney, lung, heart, stomach, adrenal, pituitary, testes, ovaries,
spleen and skeletal muscle) were conducted on all formalin-fixed control animals and the high dose animals. The remaining animals were perfused
intravenously with glutaraldehyde under sodium pentobarbital anesthesia . Quantitative assessments of teased tibial nerve preparations were
performed on all glutaraldehyde-perfused animals in control animals and the high dose animals. In addition, brain, spinal cord and sciatic nerve were evaluated microscopically (hematoxylin and eosin and Luxol-fast blue staining) from these same animals.

Other examinations:

- neurologic functions were evaluated monthly
- Parameters examined according to a scoring system: posture, gait, muscular tone, reflexes (corneal), righting and toe-pinch
- no further data

Statistics:

Body weight, hematology and clinical chemistry parameters, organ weights and organ/body weight ratios were analyzed. Mean values of
all dose groups were compared to control at each time interval.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Weekly physical examinations of all animals failed to indicate any toxic effects of the test material other than irritation at the application site. Alopecia observed on the forepaws and legs was the most common finding in both sexes; however, the incidence was spread over all test groups, including control.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

The test substance produced moderate levels of irritation, in a dose-related manner, beginning the first week of the study. Males were generally more susceptible than females to the dermal effects of the test substance throughout the study. Dermal effects (erythema and eschar formation) were only scored as present or absent therefore, the number of times per week the effects were noted was used as a general indication of the severity of the dermal observations.
- 20 mg/kg/day: erythema noted occassionally during the final 2 months; more frequently in the initial 3 weeks; exfoliation observed in approximately one-half during weeks 2 and 3 with diminishing frequency
- 66.6 mg/kg/day: erythema noted with a somewhat higher frequency than 20 mg/kg/day, frequency in females comparable to control. Exfoliation and eschar recorded for most animals by week 3.
- 200 mg/kg/day: erythema, exfoliation, and eschar seen in most animals of both sexes beginning in week 1. Atonia was observed in one males and one female, fissures present in one female and four males. A persistant fissuring was observed in one male rat from week 2 through week 7.
Males appeared somewhat more sensitive than females to erythema and eschar formation.

Mortality:

no mortality observed

Description (incidence):

All animals survived to the scheduled termination of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significantly reduced body weights in high dose male rats (versus control male rats) were noted from weeks 3 through 12, except during weeks 10 and 11. Male rat weights of the low and mid dose groups were also reduced in dose-related fashion, but the differences were not statistically significant in comparison to control values. No statistically significant effects on weight were seen in treated female rats, however, high dose female animals never exceeded 95% of the mean weights of control females after 2 weeks on test.

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:

no effects observed

Description (incidence and severity):

None of the hematologic parameters evaluated differed significantly from control values.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Of the clinical chemical parameters which were determined at the study termination, none were affected in a manner suggestive of a treatment-effect.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Protein (2+, 100 mg/dl) was confirmed to be present in the urine of one mid-dose male and female, in two high-dose males and one high-dose female. A large amount of occult blood was also present in the urine of this one high-dose female. The specific gravity of this high-dose female and one mid-dose female was also high (>1.090). These findings suggested a possible effect of the test substance on the kidneys, but no alterations were observed in the histopathological examination to support this conclusion. Additionally, without relation to urinary volumes and creatinine, the relevance of in urinary protein concentration is questionable. All in all, the singular changes are not considered adverse by the registrant.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was a significant downward trend in male liver weights; however, this was not evident in the liver/body weight ratio and is therefore of doubtful significance. Other organ weights and organ/body weight ratios were comparable across all groups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Formalin-fixed rats:
No changes, gross or microscopic, were evident which could be attributed to a systemic toxic effect of the test substance. The most common spontaneous gross necropsy findings, occurring across all groups, were inflammations around the ear tags, and slight hair loss on the extremities. No unusual microscopic pathological findings were evident which could be attributed to the topical administration of the test substance.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Month 1 neurological function tests showed two high-dose males with slightly reduced corneal response. All other evaluations were normal.
Month 2 neurological function tests were unremarkable in control, low and mid dose animals (both sexes). Four high dose males and three high dose females showed slightly abnormal gait described as "stilted". A slight decreased corneal reflex was observed in four males and one female. A moderately decreased toe pinch response (hindtoes only) was also present in one male rat.
Month 3 neurological function tests showed a slightly stilted gait and altered righting reflex in one male control rat and a slightly relaxed body tone in one mid dose male rat. One male high dose animal continued to exhibit a moderately decreased toe-pinch response (hindtoes only) . All neurological observations were normal in both the control and treated female rats at Month 3.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Glutaraldehyde-perfused rats:
Histopathological examinations of hematoxylin and eosin and Luxol-fast Blue stained slides of brain, spinal cord and sciatic nerve from ten rats (5/sex) treated with the high dose of the test substance failed to reveal any treatment-related lesions when these tissues were compared to similar ones from ten control rats (5/sex). In addition, microscopic examination of 50 teased nerve fibers from the tibial nerve of ten high-dose animals (5/sex) were comparable to those of the controls. When quantitative measurements were taken of myelinated nerve fibers in cross-section of the distal sciatic nerve, a slight shift to larger diameters could be detected in high-dose males when compared to the controls. However, there was also a slight decrease in fiber diameters in treated females. The relatively large standard deviation in data from both males and females suggest that these slight changes in fiber diameters are not significant. Moreover, the absence of lesions by more conventional histopathological examinations substantiate this conclusion.

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

LOAEL

Remarks:

skin irritation

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

dermal irritation

Dose descriptor:

NOAEL

Remarks:

for systemic toxicity

Effect level:

66.66 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Dose descriptor:

NOAEL

Remarks:

for systemic toxicity

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects observed

Critical effects observed:

yes

Lowest effective dose / conc.:

20 mg/kg bw/day (nominal)

System:

integumentary

Organ:

skin

Treatment related:

yes

Dose response relationship:

yes

Conclusions:

The test substance did not induce neurotoxicity and no sub-chronic toxicity other than dermal irritation and decreased body weight was observed in animals, under the conditions tested. Thus, the NOAEL for local effects was considered to be 20 mg/kg/day and the NOAEL for systemic effects (based on body weight) was concluded to be 66.6 mg/kg/day.

Executive summary:

The sub-chronic dermal application of the test substance to the backs of Sprague-Dawley rats was studied to assess potential neurotoxic and other local and systemic toxic effects. Three groups of 20 rats each (10/sex/level) were treated topically with 20, 66 2/3, and 200 mg/kg/day 5 days per week for 3 months. Twenty control animals (10/sex) were treated with corn oil. Solutions at appropriate levels were prepared in corn oil and a constant dose volume (2.077 ml/kg) was applied to all animals. Treatment sites were not occluded. Dermal observations were performed pretest and 5 times/week throughout the study. Clinical laboratory studies were performed at termination. Neurological function evaluations were performed at months 1, 2, and 3. At three months, all animals were terminated. One-half of the animals were sacrificed by exsanguination under light ether anesthesia, and selected organs and tissues were fixed in formalin. Organ and organ body weight ratios were determined on all of these animals only. Histopathological evaluations of 10 organs or tissues were conducted on all formalin-fixed Group 1 and 4 animals. The remaining animals were perfused intravenously with glutaraldehyde under sodium pentobarbital aesthesia. Quantitative assessments of teased tibial nerve preparations were performed on all glutaraldehyde-perfused animals in Group 1 and 4. In addition, brain, spinal cord and sciatic nerve were evaluated microscopically (hematoxylin and eosin and Luxol-fast Blue staining) from these same animals. Erythema, eschar and exfoliation were recorded during the initial week of the study and maximum frequency of these effects were seen during Weeks 2 and 3. A dose-response was noted, with males being slightly more susceptible. In the last 2 months of the study most treated animals developed an apparent tolerance to the irritant effects of the test substance. Significantly lower body weights were recorded throughout the study for Group IV males (200 mg/kg/day). Body weights for female rats and Group II and III male rats were not significantly affected. Routine toxicologic and pharmacologic signs were unremarkable throughout the study. Month 2 neurologic evaluations showed an effect on gait in 4 of 10 male and 3 of 10 female Group IV rats. Reduced corneal reflex was also seen in some rats in this group; however, Month 3 evaluations failed to show these effects. Hematological and clinical chemistry parameters appeared unaffected by treatment with the test substance. However, there was a dose-related increase in urinary protein values in both sexes. Organ weights, gross necropsy observations and microscopic studies did not reveal any systemic toxic effects. Using tibial nerve teasing techniques, no morphometric differences were found between Group land Group IV glutaraldehyde perfused rats. Quantitative assessment of tibial nerve fiber cross-sections showed slightly increased diameters for males and slightly decreased diameters for females. These changes were not considered to be treatment-related.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

66.66 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

OECD TG 411 study

System:

integumentary

Organ:

skin

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Dermal:

There are no data available to assess the toxicity of DPGDA after dermal repeated dose administration.

However, there are valid data available which assessed the dermal toxicity of the structurally related tripropylene glycol diacrylate (Cas No. 42978-66-5) after repeated dose administration (Endpoint study record referenced in chapter 7.9.1. Neurotoxicity). These data were adopted from tripropylene glycol diacrylate for DPGDA by read-across:

 

The subchronic dermal application of the test material tripropylene glycol diacrylate (TPGDA) to the backs of Sprague-Dawley rats was studied to assess potential neurotoxic and other local and systemic effects (Bio/Dynamics Inc. 1982, Val. 2). Ten male and female Sprague-Dawley rats per dose were exposed on their backs to doses of 0, 20, 66.66, 200 mg/kg bw/d, 5 days a week for 90 days. The animals were observed for mortality and gross signs of toxicologic or pharmacologic effects, and the body weight was determined weekly. Blood was obtained, haematology, clinical chemistry and urinalysis were conducted. Neurologic functions were evaluated monthly (posture, gait, muscular tone, corneal reflexes, righting and toe-pinch). One-half of the animals were sacrificed by exsanguination under light ether anaesthesia, and selected organs and tissues were fixed in formalin. Organ and organ-body weight ratios (adrenals, brain, liver, kidney, heart, spleen, testes with epididymides, ovaries) were determined on all of these animals only. Histopathological evaluations of 10 organs or tissues (liver, kidney, lung, heart, stomach, adrenal, pituitary, testes, ovaries, spleen and skeletal muscle) were conducted on all formalin-fixed control animals and the high dose animals. The remaining animals were perfused intravenously with glutaraldehyde under sodium pentobarbital anaesthesia . Quantitative assessments of teased tibial nerve preparations were performed on all glutaraldehyde-perfused animals in control animals and the high dose animals. In addition, brain, spinal cord and sciatic nerve were evaluated microscopically (hematoxylin and eosin and Luxol-fast blue staining) from these same animals.

All animals survived to the scheduled termination of the study; weekly physical examinations of all animals failed to indicate any toxic effects of the test material other than irritation at the application site. Alopecia observed on the forepaws and legs was the most common finding in both sexes; however, the incidence was spread over all test groups, including control. The test substance produced moderate levels of irritation, in a dose-related manner, beginning the first week of the study . Males were generally more susceptible than females to the dermal effects of the test substance throughout the study. Dermal effects (erythema and eschar formation) were only scored as present or absent therefore, the number of times per week the effects were noted was used as a general indication of the severity of the dermal observations.

Dermal effects were absent in the controls except for one female rat which had one exfoliation score in Week 8, and one other female rat which exhibited eschar and exfoliation at various times during the initial 2 months of the study. In the low dose group, erythema was only noted occasionally during the final 2 months of the study. In the initial 3 weeks, erythema was recorded more frequently (approximately 12-13 % of total observations) than later in the study. Exfoliation was recorded with gradually diminishing frequency in approximately one-half of the low dose group animals (both sexes) during weeks 2 and 3. During the last 5 weeks of the study, only two female rats and one male rat were observed with this effect. Oedema, atonia, and fissuring were not observed in any low dose group animals. In mid dose group male rats, erythema was noted with a same what higher frequency than was seen for low dose group male rats. Erythema frequency for female mid dose group rats was comparable to low dose group. After week 3 of the study, a somewhat lower frequency of erythema was recorded in both sexes. Exfoliation and eschar were recorded for most animals in the mid dose group by week 3 of the study, with diminishing frequency thereafter. As in the low dose group rats, oedema, atonia and fissuring were not observed. In the high dose group, erythema, exfoliation and eschar were seen in most animals of both sexes beginning in week 1. The highest frequency was noted in week 2 (both sexes), with diminishing frequency thereafter. Atonia was observed in one male and one female during weeks 3-4 and 5-6, respectively. Fissures were present in one female rat (on one day during week 2 only). Four male rats showed fissures on week 2 of the study. A persistent fissuring in one of these rats was observed from week 2 through week 7 of the study. Male rats appeared somewhat more sensitive than females to erythema and eschar formation.

Significantly reduced body weights in high dose male rats (versus control male rats) were noted from weeks 3 through 12, except during weeks 10 and 11. Male rat weights of the low and mid dose groups were also reduced in dose-related fashion, but the differences were not statistically significant in comparison to control values. No statistically significant effects on weight were seen in treated female rats, however, high dose female animals never exceeded 95% of the mean weights of control females after 2 weeks on test.

Of the clinical chemical parameters which were determined at the study termination, none were affected in a manner suggestive of a treatment-effect.

None of the hematologic parameters evaluated differed significantly from control values. Protein (100 mg/dl) was confirmed to be present in the urine of one mid-dose male and female, in two high-dose males and one high-dose female. A large amount of occult blood was also present in the urine of this one high-dose female. The specific gravity of this high-dose female and one mid-dose female was also high (>1.090). These findings suggested a possible effect of the test substance on the kidneys. However, histopathology was comparable to controls.

There was a significant downward trend in male liver weights; however, this was not evident in the liver/body weight ratio and is therefore of doubtful significance. Other organ weights and organ/body weight ratios were comparable across all groups.

Month 1 neurological function tests showed two high-dose males with slightly reduced corneal response. All other evaluations were normal. Month 2 neurological function tests were unremarkable in control, low and mid dose animals (both sexes). Four high dose males and three high dose females showed slightly abnormal gait described as "stilted". A slight decreased corneal reflex was observed in four males and one female. A moderately decreased toe pinch response (hindtoes only) was also present in one male rat. Neurological function tests showed a slightly stilted gait and altered righting reflex in one male control rat and a slightly relaxed body tone in one mid dose male rat. One male high dose animal continued to exhibit a moderately decreased toe-pinch response (hindtoes only). All neurological observations were normal in both the control and treated female rats at Month 3.

No changes, gross or microscopic, were evident which could be attributed to a systemic toxic effect of the test substance in formalin-fixed rats: The most common spontaneous gross necropsy findings, occurring across all groups, were inflammations around the ear tags, and slight hair loss on the extremities. No unusual microscopic pathological findings were evident which could be attributed to the topical administration of the test substance.

Histopathological examinations of haematoxylin and eosin and Luxol-fast Blue stained slides of brain, spinal cord and sciatic nerve from ten glutaraldehyde-perfused rats (5/sex) treated with the high dose of the test substance failed to reveal any treatment-related lesions when these tissues were compared to similar ones from ten control rats (5/sex). In addition, microscopic examination of 50 teased nerve fibres from the tibial nerve of ten high-dose animals (5/sex) were comparable to those of the controls. When quantitative measurements were taken of myelinated nerve tibers in cross-section of the distal sciatic nerve, a slight shift to larger diameters could be detected in high-dose males when compared to the controls. However, there was also a slight decrease in fibre diameters in treated females. The relatively large standard deviation in data from both males and females suggest that these slight changes in fibre diameters are not significant. Moreover, the absence of lesions by more conventional histopathological examinations substantiate this conclusion.

The NOAEL for systemic effects was set here at 66.66 mg/kg bw/d due to reduced body weight only. Due to the local irritating effects observed on skin, a LOAEL local was set at 20 mg/kg bw/d.

 

Oral:

The objectives of this OECD TG 422 study performed in compliance with CLP was to determine the potential toxic effects of DPGDA. Wistar Han rats were administered the test substance dose levels of 0, 40, 125, 375 mg/kg/day, based on the results of the Dose Range Finder. With the exception of the control group, all animals (10 animals/sex/group) received the test substance orally via gavage for a minimum period of 28 days using corn oil as vehicle at a dose volume of 5 mL/kg body weight. Chemical analyses of formulations were conducted twice during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, clinical pathology, measurement of thyroid hormone T4 (parental males), gross necropsy findings, organ weights and histopathologic examinations.

Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously. Parental toxicity was observed at the highest dose level tested (375 mg/kg/day). One female at 40 mg/kg/day was sacrificed in extremis on post-coitum Day 23 because of delivery difficulties. This female delivered 3 dead pups and showed hunched posture, piloerection and pale appearance. At necropsy, 8 dead foetuses were present in the uterus. Microscopically the uterus showed characteristics of pregnancy. Findings regarded secondary to the presence of dead foetuses in the uterus were moderate multifocal hepatocellular necrosis of the centrilobular area of the liver and multifocal thrombi in the lungs. These findings in liver and lungs were attributed to the poor condition of this female. This death was regarded to be related to delivery difficulties and unrelated to the treatment with the test item. Significant body weight loss in one female at 375 mg/kg/day during the first days of lactation coincided with piloerection and a slightly pale and lean appearance. At the incidence observed, this was considered not toxicologically relevant. Body weight gain in males at 375 mg/kg/day was decreased compared with control on Day 8 of treatment, which recovered up to similar levels as control at the end of the mating period. Absolute body weights in males at 375 mg/kg/day were decreased compared with control from Day 1 of treatment up to the day of scheduled necropsy. Considering the magnitude of change and as Group 4 males just happened to be the smallest rats in this study, no toxicological relevance was attached to this finding. Relationships were suspected between gross necropsy (irregular surface of the forestomach), clinical pathology (increased neutrophil concentrations) and histopathology observations. The combination of microscopic stomach findings observed in most males and females treated at a dose of 375 mg/kg/day consisted of slight to marked squamous cell hyperplasia with slight to moderate hyperkeratosis and minimal to slight mixed cell inflammation. In addition, a forestomach ulcer at minimal degree was recorded in a single male at 125 and at 375 mg/kg/day and in a single female at 375 mg/kg/day. This combination of findings is considered adverse at the recorded incidences and severities. Mixed cell inflammation was recorded in all selected males, compared to 3/5 selected females and likely correlated to the increased neutrophils recorded for males at 375 mg/kg/day. The forestomach findings and salivation were considered to be local test item effects and likely resulting from irritating properties of the test item. The low severities of stomach findings in a single male at 125 mg/kg/day only were considered to be non-adverse. Increased mitotic figures were recorded in the liver of two females at 375 mg/kg/day and a relation to treatment with the test item could not be excluded. This alteration was considered to be non-adverse at the recorded minimal severity.

Non-adverse changes were recorded in clinical biochemistry parameters: increased cholesterol levels and increased calcium levels in females at 375 mg/kg/day. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, body weight, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), food consumption, clotting parameters, male T4 thyroid hormone levels, and organ weights). No reproductive toxicity was observed up to 375 mg/kg/day.

In conclusion, based on the results of this combined 28-Day repeated dose toxicity study the established No Observed Adverse Effect Level (NOAEL) of DPGDA (CAS 57472-68-1) was 125 mg/kg/day based on macroscopic and microscopic stomach findings and increased neutrophil counts). 

 

 

Inhalation:

No data available.

Justification for classification or non-classification

Based on the available data for repeated dose toxicity after subacute oral (gavage) and subchronic dermal administration no classification is justified.

There are currently no data available which would justify a classification of DPGDA for its repeated inhalative toxicity.