Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: OECD 422
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2010- October 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline 422 study conducted under GLP conditions at professional contract laboratory
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
KGA is 2 keto-L-gulonic acid anhydrous, which is a precursor to ascobic acid (vitamin C). KGA and Ascorbic Acid are the two primary constituents making up the multiconstituent substance KGA Greens. The remainder of KGA Greens are various other sugars and organic acids.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Indianapolis, IN
- Age at study initiation:
- Weight at study initiation: males: 244-296 g; females: 173-205 g
- Fasting period before study:
- Housing: Stainless steel, suspended, wire bottom cages for all animal during the pre-mating period. Plastic cages during mating and for females and pups after mating. Animals individually housed.
- Diet (e.g. ad libitum): PMI Feeds, Inc. Forumlab #5008, available ad libitum. Analyzed by manufacturer for nutrient content.
- Water (e.g. ad libitum): Municipal water supply analyzed by TNRCC Water Utilities Division; tap water, available ad libitum (automatic system)
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 =/- 3 degrees C
- Humidity (%): 30-70%
- Air changes (per hr): 10-12 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

IN-LIFE DATES: From: 19 April 2010 (beginning of definitive test) To: 11 June 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
The test animals were dosed by oral gavage with an appropriately sized stainless stell ball-tipped dosing needle and syringe since gavage dosing assures the proper dose presentation. Three groups (Groups II, III and IV) of ten males and ten females each were dosed daily at 500 mg/kg, 1000 mg/kg and 2000 mg/kg, respectively. A concurrent vehicle control group (Group I, ten males and ten females) was dosed with the vehicle, deionized water. Groups were dosed daily at approximately the same time each day and dose amounts were adjusted weekly based on the latest body weight. All animals were dosed at a constant volume of 8.0 mL/kg. Males and females were dosed for 14 days prior to mating. Males were dosed for another 14 days during mating, for a total of 28 days. Females were dosed through mating and until one day prior to termination (lactation Day 4 for those that delivered and Study Day 53 for those that did not deliver).
Details on mating procedure:
- M/F ratio per cage: one female was cohabited with one male
- Length of cohabitation: two weeks or until signs of pregnancy were observed
- Proof of pregnancy: presence of either a vaginal plug or sperm in vaginal smear is counted as gestation day 0 of pregnancy
- After successful mating each pregnant female was put in plastic cages and nesting materials were added
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose concentrations were verified weekly. One sample for analysis was taken from the center of each dosing formulation prepared weekly during the in-life phase of the study. The mean concentrations for the Group II-500 mg/kg, Group III-1000 mg/kg, and Group IV-2000 mg/kg solutions were 67.5 mg/mL, 134.6 mg/mL, and 267.2 mg/mL, respectively. On average, males from Groups I-IV were dosed with 2.4, 2.3, 2.3 and 2.4 mg/mL of test substance, respectively. Females from Groups I-IV were dosed with an average of 1.8 mg/mL of test substance.
Duration of treatment / exposure:
Males and females were dosed for 14 days prior to mating. Males were dosed for another 14 days during mating, for a total of 28 days. Females were dosed through mating and until one day prior to termination (lactation Day 4 for those that delivered and Study Day 53 for those that did not deliver).
Frequency of treatment:
Daily as described above
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 500, 1000, 2000 mg/kg daily
Basis:
actual ingested
No. of animals per sex per dose:
Ten males and ten females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a range finder test and a pretest
- Rationale for animal assignment (if not random): Random

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for males. Body weights for females were recorded on Days 7 and 14, and on Gestation Days 0, 4, 7, 11, 14, 17 and 20, and on Lactation Days 1 and 4. Body weights of pups were recorded on Lactation Days 1 and 4. Body weights were also recorded at the time of discovery for animals that died during the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, food consumption was recorded weekly through the termination of the study with the exception of the cohabitation period.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable
- Time schedule for examinations: Not applicable
Litter observations:
Parturition and Litter Observations: The day parturition was complete was considered Lactation Day 0. When birth was completed, the litters were sexed, examined for gross malformations, and the number of stillbirths and live pups was recorded. The pups were individually marked for identification. Any changes or abnormalities in nesting or nursing behavior were recorded. Body weights for the pups were recorded on Lactation Days 1 and 4, and they were re-sexed at that time. Pups were observed daily for general appearance, behavior and survival.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed and evaluated on day 28.
- Maternal animals: All surviving animals were sacrificed and examined on lactation day 4 or for those not giving birth, study day 53.

GROSS NECROPSY/HISTOPATHOLOGY/ORGAN WEIGHTS
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]A gross necropsy examination was conducted on each adult animal at the time of discovery after death or at time of sacrifice. The necropsy included gross observations of external surfaces and all orifices, and gross observations of thoracic and abdominal cavities and their viscera. Special attention was paid to the organs of the reproductive system. The number of corpora lutea was recorded. The testes and epididymides of all sacrificed males and ovaries with oviducts of the sacrificed females were carefully removed, trimmed, and weighed. Ovaries with oviducts, testes, epididymides and accessory sex organs, and all organs showing macroscopic lesions were preserved. Tissues were weighed as soon as possible to avoid dehydration of the tissues. The liver, kidneys, thymus, spleen, brain and heart from five adult males and females randomly selected from each group were carefully removed, trimmed and weighed. The following tissues were preserved in 10% neutral buffered formalin: all gross lesions or tissues showing abnormalities, brain (cerebrum, cerebellum and pons), spinal cord, stomach, small and large intestine (duodenum, jejunum, ileum, caecum and colon including Peyer’s patches), liver (sections of 2 lobes), kidneys, adrenals, spleen, heart and aorta, thymus, thyroid, trachea and lungs (including bronchi, preserved by inflation with fixative and then immersion), ovaries with oviducts, uterus, urinary bladder, lymph nodes (mesenteric and submandibular), peripheral nerve (sciatic or tibial) in close proximity to the muscle, and bone marrow.

A full histopathologic examination on all tissues saved from five males and five females from the control (Group I) and high-dose (Group II) groups and on all tissues saved from any animal which died on study. Special emphasis was put on the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Observations for morbidity and mortality were made twice daily, and observations for pharmacologic and/or toxicologic effects were made once daily until study termination. General clinical observations included but were not necessarily limited to evaluation of skin, fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects (salivation, lacrimation, excessive urination and diarrhea), central nervous system effects (tremors and convulsions), changes in the level of activity, gait and posture, reactivity to handling or sensory stimuli, altered strength and stereotypies or bizarre behavior (e.g., self mutilation, walking backwards). The nature, onset, severity, and duration of all gross or visible pharmacologic and/or toxicologic effects were recorded. Detailed clinical observations were made weekly in the same manner as pretest.
Statistics:
The findings were evaluated in terms of the observed effects, necropsy, and microscopic findings. The evaluation includes the relationship between the dose of the test substance and the presence or absence, incidence and severity of abnormalities, including gross lesions, identified target organs, infertility, clinical abnormalities, affected reproductive and litter performance, body weight changes, effects on mortality and any other toxic effects. Accepted statistical methods were employed where appropriate, including one-way analysis of variance (ANOVA) and Dunnett’s t-test to identify differences from control when identified by ANOVA.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Seven animals, all but one in 2000 mg/kg dose
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Sporadic
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Sporadic
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): Seven animals, 2 males and 5 females, died during the study. All but one animal was from Group IV-2000mg/kg. It is believed that the animal from Group II-500 mg/kg died because of a technician error during dosing. The animals in Group IV-2000 mg/kg tended to struggle and resist more during dosing than animals from the lower dose groups. It can be surmised that the test substance may have been causing some irritation or inflammation at this dosage level causing increased resistance to dosing which in turn caused aspiration of gavage material. Histopathology results confirmed that the likely cause of death for all these animals was aspiration from the gavaged material. The pathology report speculates that the aspiration of gavage material may be a result of technician error or that the test substance was so distasteful that the animal gags or attempts to regurgitate and thus aspiration occurs.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): On Day 7, Group IV-2000 mg/kg male body weights were significantly lighter than Group I-Control (p=0.0435) and on Day 28, Group II-500 mg/kg male body weights were significantly lighter than Group III-1000 mg/kg rats (p=0.0191). There were no other significant differences between the groups for the male body weights or the body weight changes. For the female body weights, on Gestation Day 20 and Lactation Day 1, the body weights of the para females in Group III were significantly lighter than those of the Group I rats (p=0.0515 and 0.0086). Group IV nulliparous females had significantly less body weight change from the presumed Gestation Day 0 to Study Day 53 than Group I nulliparous females (p=0.0013). There were no other significant differences between groups for female body weights or body weight changes.

Among male food consumption amounts, from Days 7 through Day 14, animals from Group II-500 mg/kg consumed significantly more food than animals in Group IV-2000 mg/kg (p=0.0379). There were no other significant differences among food consumption amounts for males between groups. Among female food consumption amounts, rats in Group IV-2000 mg/kg consumed significantly less food for Days 0-14 than animals in Group I-Control (p=0.0103 and 0.0391). There were no other significant differences among food consumption amounts for females between groups.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): Dose concentrations were verified weekly. One sample for analysis was taken from the center of each dosing formulation prepared weekly during the in-life phase of the study. The mean concentrations for the Group II-500 mg/kg, Group III-1000 mg/kg, and Group IV-2000 mg/kg solutions were 67.5 mg/mL, 134.6 mg/mL, and 267.2 mg/mL, respectively. On average, males from Groups I-IV were dosed with 2.4, 2.3, 2.3 and 2.4 mg/mL of test substance, respectively. Females from Groups I-IV were dosed with an average of 1.8 mg/mL of test substance.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): There were 4, 4, 7 and 3 females in Groups I – IV, respectively, that had successful births. Little difference is apparent in conception rates, number of pups delivered or incidence of stillborn pups when control and high-, mid- and low-dose groups are compared. Corpora lutea counts at necropsy are presented in Table 14. There were no significant differences in corpora lutea counts among groups.

ORGAN WEIGHTS (PARENTAL ANIMALS): There were no statistically significant differences among groups in weights of male organs. For females, there were no statistically significant differences among groups in the weights of female organs with one exception. The adrenal glands of Group III-1000 mg/kg females were significantly lighter than the adrenal glands of Group IV-2000 mg/kg (p=0.0311).

GROSS PATHOLOGY (PARENTAL ANIMALS): For males that were terminally sacrificed on schedule (Day 29), the only findings related to mottled kidneys found in nine animals among all four groups and one animal in Group II-500 mg/kg with an abnormally small right testicle. For the two males from Group IV-2000 mg/kg that died during the study, both had extreme gas in the stomach and one had discolored lymph nodes, matted hair around muzzle, gas in the intestines and expanded stomach dermal tissue. For para females that were sacrificed on schedule (Lactation Day 4), there were no observable abnormalities for any animal in any group. For nulliparous females that were sacrificed on schedule (Study Day 53), one animal had a flattened dorsal kidney, one animal had a flattened dorsal kidney and uterine horns filled with fluid and one animal had uterine horns filled with fluid. For the five females that died on study, abnormal findings included discolored lungs, liver and spleen, and the contents of the stomach, chest cavity, mouth, uterus, and small intestine.

HISTOPATHOLOGY (PARENTAL ANIMALS): The histopathology report concluded “No histologic test article related lesions were observed in this study. The reproductive organs of treated rats were similar to those of control rats. Several treated rats that died on study were highly suspect of aspiration of gavage material (two Group 4 males, three Group 4 females, and one Group 2 female). A number of spontaneous lesions commonly observed in rats were diagnosed. These lesions occurred in treated and control rats of both sexes and are not considered to be test article related. ” The report specified that of the rats which had died on test, “Some animals had good evidence of aspiration of the gavage material (inflammation in trachea and lungs), while others were only suspicious, but highly suspect of aspiration.”

OTHER FINDINGS (PARENTAL ANIMALS): For males, there were no significant differences between groups for any of the hematology parameters analyzed. For females, Group IV-2000 mg/kg white blood cells were significantly lower than Group II-500 mg/kg or Group III-1000 mg/kg (p=0.0020). There were no significant differences between groups for males or females for prothrombin and activated partial thromboplastin times. There were no significant differences between groups in the male serum chemistry data. For females, Group II-500 mg/kg total bilirubin was significantly lower than Group I-Control (p=0.0482). Group III potassium was significantly higher than Group II (p=0.0274). There were no other significant differences. No significant effects were observed in the functional observational battery or motor activity.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed

Effect levels (P1)

Remarks on result:
not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING): Little difference is apparent in conception rates, number of pups delivered or incidence of stillborn pups when control and high-, mid- and low-dose groups are compared. There were no significant differences in corpora lutea counts among groups.

CLINICAL SIGNS (OFFSPRING): Daily observations of the litters revealed no observable abnormalities except for instances of pups not found or being found dead. On Lactation Day 1, one pup in Group III-1000 mg/kg was found thin and pale.

BODY WEIGHT (OFFSPRING): For Lactation Day 1 mean litter weights, Group III-1000 mg/kg was significantly lighter than any of the other groups and Group IV-2000 mg/kg weights were significantly heavier than Group I-Control (p=0.0001). For Lactation Day 4 mean litter weights, Group IV was significantly heavier than any of the other groups (p=0.0001).

Effect levels (F1)

Remarks on result:
not determinable due to absence of adverse toxic effects

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
no effects observed

Developmental neurotoxicity (F2)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
no effects observed

Effect levels (F2)

Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Parturition and Litter Observations: Group birthing information is summarized below.

Group

% Conception

% Successful

Mean #

Mean Live

Mean

Delivery

Pups

Pups

Stillborn

 

 

I-Control

40.0%

100%

16

16

0

II-500 mg/kg

44.4%

100%

15

15

0

III-1000 mg/kg

70.0%

100%

15

14

1

IV-2000 mg/kg

57.1%

75%*

14

14

0

Group means for individual pup body weights on Lactation Days 1 and 4 are summarized below:

Group

Lactation Day 1 Means

Lactation Day 4 Means

Gain

I-Control

92.7

132.2

39.5

II-500 mg/kg

94.9

123.0

28.0

III-1000 mg/kg

82.6

121.9

39.3

IV-2000 mg/kg

95.3

146.2

50.9

Weights are in grams

 

 

 

A summary table of the reproductive measurements is shown below:

Observation

Group I
Control

Group II
500 mg/kg

Group III
1000 mg/kg

Group IV
2000 mg/kg

Pairs started (N)

10

9

10

8

Females showing evidence of copulation (N)

6

9

10

7

Females achieving pregnancy (N)

4

4

7

3

Conceiving days 1 – 5 (N)

4

7

8

6

Conceiving days 6 – 14 (N)

2

2

2

1

Dams with live young born (N)

4

4

7

3

Dams with live young at Day 4 (N)

4

4

7

3

Corpora lutea/dam (mean)

22.25

18.25

18.14

15.33

Live pups/dam at birth (mean)

16

15

15

14

Live pups/dam at Day 4 (mean)

15

14

13

14

Sex ratio (m/f) at birth (mean)

0.63

0.97

0.59

0.84

Sex ratio (m/f) at Day 4 (mean)

0.81*

0.88

0.79*

0.94

Litter weight at birth (mean)

92.7

94.9

82.6

95.3

Litter weight at Day 4 (mean)

132.2

123.0

121.9

146.2

Pup weight at birth (mean)

6.4

6.5

6.2

6.8

Pup weight at Day 4 (mean)

9.1

8.7

9.3

10.5

Dams with abnormal pups

None

None

None

None

(N) - Number; m - Male; f - Female; * - mean includes one litter sexed on Day 1 instead of Day 4

Applicant's summary and conclusion

Conclusions:
No effects on either male or female reproductive performance was observed. Based on body weight gain reductions in the 2000 mg/kg bw/day group, the NOAEL was determined to be 1000 mg/kg bw/day.
Executive summary:

The OECD 422 combined repeated dose study with reproductive and developmental toxicity screening was conducted to determine the subchronic toxicity to rats. Groups of rats were exposed by oral gavage to doses of 0, 500, 1000 and 2000 mg/kg bw/day of KGA Greens. Males were exposed for 28 days and females were exposed up through lactation day 4 or 53 days, after which surviving rats were sacrificed and necropsied. Observations of mortality and a suite of other examinations were conducted. Results show sporadic effects on various endpoints in the 2000 mg/kg bw/day high dose. At this 2000 mg/kg bw/day level the test substance appeared to be so irritating or distasteful that the animals tended to struggle and/or resist dosing, which in turn led to deaths likely caused by aspiration of gavaged material. No significant dose-related effects were observed in the 500 and 1000 mg/kg bw/day doses. No significant effects was observed at any dose on male or female reproductive performance. The NOAEL was determined to be 1000 mg/kg bw/day based on sporadic weight gain reductions in the maternal generation.