Desmedipham

Specific investigations: other studies

Link to relevant study record(s)

Reference

Endpoint:

mechanistic studies

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2013-03-12 to 2014-06-03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Non-guideline mechanistic study

Qualifier:

according to guideline

Guideline:

other: US EPA OCSPP 870.SUPP

Deviations:

not specified

GLP compliance:

no

Type of method:

in vivo

Endpoint addressed:

repeated dose toxicity: oral

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Species: Rat
Strain: Wistar
Age: 10 weeks
Weight at dosing: 361-413 g
Acclimation period: 14 days
Diet: Irradiated diet A04CP1-10
Water: Softened tap water from municipal supply
Housing: Suspended polycarbonate wire-mesh cages
Environmental conditions
Temperature: 20-24 °C
Humidity: 40-70 %
Air changes: 10-15
Photoperiod: 12 hours

Animal assignment and treatment: A total of 45 male rats out of 50 were selected for the study conduct and assigned to the study groups.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

The test item was incorporated into the diet by dry mixing to provide the required dietary concentrations. When not in use, the diet formulations were stored at approximately -18°C. There were four formulations for each concentration for the study.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days

Frequency of treatment:

Continuous

Post exposure period:

2-3

Dose / conc.:

0 ppm

Dose / conc.:

1 500 ppm

Dose / conc.:

4 000 ppm

No. of animals per sex per dose:

15

Control animals:

other:

Examinations:

1. Observations:

All animals were checked for moribundity and mortality twice daily (once daily on weekends or public holidays). All animals were observed for clinical signs at least once daily. Detailed physical examinations were performed at least weekly during the treatment period. The nature, onset, severity, reversibility, and duration of clinical signs were recorded. Cages and cage-trays were inspected daily for evidence of ill-health, such as blood or loose feces.

2. Body weights:

Each animal was weighed at least weekly during the acclimatization period, on the first day of test item administration, then at weekly intervals throughout the treatment period. Additionally, animals were weighed before scheduled necropsy (terminal body weight).

3. Food consumption and compound intake:

The weight of food supplied and that remaining at the end of the food consumption period was recorded weekly for all animals during the treatment period. Any food spillage was noted. The weekly mean achieved dosage intake in mg/kg bw/day for each week and for Weeks 1 to 4 (average of the 4 weeks period) was calculated for each sex.


4. Clinical pathology:

Blood sampling: On Study Day 30 and 31, blood samples were taken from all animals in all groups by puncture of the retro-orbital venous plexus. Animals were anesthetized by inhalation of Isoflurane (Virbac, Carros, France). Blood was collected on lithium heparin and centrifuged for hormone level investigation.

Hormone investigations:
Plasma was prepared from each blood sample and kept frozen at approximately -74°C until the determination of T3, T4 and TSH hormone levels with specific ELISA kits (supplied by Calbiotech, Spring Valley, CA 91978, USA, for T3 and T4 and by BlueGene, Putuo District, Shanghai, China, for TSH).

5. Post mortem examinations:

On Study Day 30 and 31, all animals from all groups were sacrificed by exsanguination while under deep anesthesia (Isoflurane inhalation).
All animals were necropsied. The necropsy included the examination of all major organs, tissues and body cavities. Macroscopic abnormalities were recorded, sampled and examined microscopically.

At final sacrifice the following organs were weighed: Brain, liver,
Tissue collection: Liver, pituitary gland, thyroid gland (with parathyroid gland), macroscopic findings.

For each group, two pieces of both the median and the left lobe of the liver plus the pituitary gland from each animal were collected and flash frozen in liquid nitrogen. These samples were stored at approximately -74°C until used for Q-PCR investigations. In addition, the thyroid gland was collected, flash frozen in liquid nitrogen and stored at approximately -74 °C for possible further analysis.

6. Hepatotoxicity testing:

Performed at CXR Biosciences Ltd. (CXR with the exception of the microsome preparation. At final necropsy, the remaining portions of the liver from all animals were homogenized for microsomal preparations. Microsomes were sent to CXR in order to determine total cytochrome P-450 content, specific cytochrome P-450 isoenzyme profile and glucuronidation activity (T4-GT) to check the hepatotoxic potential of the test item.
Total microsomal cytochrome P-450 content: The microsomal total P-450 content was determined spectrophotometrically, as described by Omura and Sato (1964), according to LMS Spec-002. Results were maintained in the Study File.
Enzymatic activities: Microsomal pentoxyresorufin-O-depentylation, used as a marker for cytochrome P450 2B activity, will be determined spectrofluorometrically by the formation of resorufin from pentoxyresorufin, as described by Burke et al (1985), according to CXR LMS Fluor-002, and
EQ-001. Results were maintained in the Study File.
Microsomal benzyloxyresorufin-O-debenzylation, used as a marker for CYP2B/3A activity, was determined spectrofluorometrically by the formation of resorufin from benzyloxyresorufin, as described by Burke et al (1985), according to CXR LMS Fluor-002, and EQ-001. Results were maintained in the Study File.
Rat liver microsomes were incubated with 125I-thyroxine and the formation of T4-glucuronide was determined by HPLC with radioflow detection.

Total RNA purification: Total RNA from liver and pituitary gland were isolated from all individual control and treated animals using RNeasy Mini kits (Qiagen). RNA was quantified and controlled using the spectrophotometer Nano Drop (Agilent Technologies). RNA quality controls were performed based on the ribosomal RNA electrophoretic profiles using a Bioanalyser (Agilent Technologies).

Quantitative PCR: An appropriate quantity of total RNA was used for Reverse Transcription (RT) using a High Capacity cDNA Archive kit (Applied Biosystems). The assay was performed in duplicate using Taqman assays (Assay on demand, Applied Biosystems), diluted first strand cDNA, FastStart Universal Probe Master Mix (Roche) on an ABI prism 7900 HT machine (Applied Biosystems).
ß-microglobulin and ß-actin were selected as reference gene for the quantitative calculations of transcripts in the liver and the pituitary gland, respectively.
Milli-Q water was used as negative control.

Details on results:

1. Observations:

There were no clinical signs and no mortalities during the study. There were no clinical signs except for one animal was noted to have hair loss on both forepaws on Study Day 31. This clinical sign was considered to be incidental.

2. Body weight, food consumption and test compound intake:

Body weight parameters were unaffected by treatment throughout the study compared to the controls. Food consumption parameters were unaffected by treatment throughout the study compared to the controls.

3. Hepatotoxicity:

At 4000 ppm increased microsomal total cytochrome P450 content by 1.2-fold, microsomal T4-GT activity by 1.3-fold and microsomal PROD activity by 1.7-fold were determined.
At 1500 ppm increased microsomal PROD activity by 1.3-fold in male Wister rats was seen. No significant effects on BROD activity were seen following administration

4. Q-PCR results:

At 4000 ppm the following gene transcripts were affected:
Liver:
Cyp2b1 gene transcripts were up-regulated (+956%, p=0.01) in the liver of desmedipham treated male rat when compared to the controls.
Cyp4a1 gene transcripts were up-regulated (+309%, p=0.01) in the liver of desmedipham treated male rat when compared to the controls.
Ugt1a6 gene transcripts were up-regulated (+71%, p=0.01) in the liver of desmedipham treated male rat when compared to the controls.
Ugt2b1 gene transcripts were up-regulated (+87%, p=0.05) in the liver of desmedipham treated male rat when compared to the controls.
Ephx1 gene transcripts were up-regulated (+55%, p=0.01) in the liver of desmedipham treated male rat when compared to the controls.
Pituitary gland:
Tshb gene transcripts were slightly up-regulated (+22%, not statistically significant) in the pituitary gland of desmedipham treated male rat when compared to the controls.

5. Terminal body weight and organ weights
At terminal sacrifice, there were no changes in mean terminal body weights between control and treated groups.
At 4000 ppm, mean relative liver weight (organ to terminal body weight ratio) was statistically significantly higher (+7%, p<0.05) when compared to the controls. This change was considered as treatment related.

Test compound intake

Mean achieved dietary intake of desmedipham (weeks 1-4)
Diet concentration (ppm)	Males (mg/kg bw/day)
1500	94
4000	252

 

Hepatotoxicity tests (performed at CXR)

Paramenter	Control 0 ppm	Desmedipham 1500 ppm	Desmedipham 4000 ppm
Total P 450 nmol/mg	0.70 ± 0.16^a	0.75± 0.21	0.86 ± 0.13**
protein	(100.0 ± 22.3)	(108.1 ±30.3)	(123.7 ± 18.6)
T4-GT
pmol T4 glucuronide	2.20 ± 0.33	2.50 ± 0.75	2.78 ± 0.70**
formed/min/mg	(100.0 ± 15.0)	(113.4 ± 34.0)	(126.1 ± 32.0)
protein
PROD
pmol resorufin	2.11 ± 0.71	2.78 ± 0.65*	3.61 ± 1.20***
formed/min/mg	(100.0 ± 33.6)	(132.2 ± 30.8)	(171.3 ± 57.0)
protein
BROD
pmol resorufin	23.42 ± 8.46	23.37 ± 5.76	24.90 ± 6.49
formed/min/mg	(100.0 ± 36.1)	(99.8 ± 24.6)	(106.3 ± 27.7)
protein

^a Values are mean± SD. Values in parenthesis are mean % control ± SD. N = 15 per group. A Student's t-test (2-sided) was performed on the results; * statistically different from control p< 0.05; **p< 0.01; ***p< 0.001.

 

Q-PCR results in the liver

	Mean Relative Quantity ± standard deviation of gene transcripts (%change compared to control mean values)
Gene transcripts	Control	Desmedipham 1500 ppm	Desmedipham 4000 ppm
Cyp1a1	0.21 ± 0.236	0.80 ± 1.435	0.51 ± 0.647
Cyp2b1	0.73 ± 0.547	2.51** ± 2.250 (+244%)	7.71** ± 6.893 (+956%)
Cyp3a3	0.87 ± 0.249	1.04 ± 0.332	1.01 ± 0.270
Cyp4a1	0.88 ± 0.325	1.48** ± 0.511 (+68%)	3.60** ± 1.262 (+309%)
Ugt1a6	0.75 ± 0.243	1.16** ± 0.355 (+55%)	1.28** ± 0.318 (+71%)
Ugt2b1	0.39 ± 0.320	0.53 ± 0.248 (+36%)	0.73* ± 0.429 (+87%)
Sult2a2	0.74 ± 0.388	0.81 ± 0.420	0.91 ± 0.507
Ephx1	0.85 ± 0.169	1.11** ± 0.297 (+31%)	1.32** ± 0.377 (+55%)
Gstm4	0.77 ± 0.172	0.83 ± 0.154	0.82 ± 0.118
Por	1.27 ± 0.494	1.51 ± 1.051	1.42 ± 0.482

 

 

Q-PCR results in the pituitary gland

	Mean Relative Quantity ± standard deviation of gene transcripts (%change compared to control mean values)
Gene transcripts	Control	Desmedipham 1500 ppm	Desmedipham 4000 ppm
Tshb	1.12 ± 0.616	1.37 ± 0.751 (+22%)	1.55 ± 0.869 (+38%)

*:P≤ 0.05; **P≤ 0.01

 

Conclusions:

This study was not performed according to any OECD guideline on male rats. Only brain and liver were weighed, not the thyroid. Liver enzyme induction can perhaps explain some of the thyroid effects but it can not exclude the presence of other MoA for thyroid effects. Hence, the question of MoA is not considered to be explained thoroughly. Desmedipham treatment up-regulates Ugt2b1 transcript from the UDP glucuronosyltransferase gene family in rats.

Executive summary:

A special study with desmedipham was conducted to evaluate the mode of action for thyroid findings which were seen in the toxicological studies. Desmedipham, an herbicide in the evaluation phase, was administered continuously via the diet to groups of male Wistar rats (15/group) for at least 28 days at concentrations of 1500 and 4000 ppm, corresponding to 94 and 252 mg/kg bw/day. One sex was sufficient since the mechanism works in both sexes. A similarly constituted group received untreated diet and acted as a control.

At 4000 ppm mean relative liver weight was higher than that of the controls. Total cytochrome P-450 content was increased by 1.2-fold, T4-GT activity by 1.3-fold and PROD activity by 1.7-fold.

In the liver, the up-regulated phase I enzyme gene transcripts were Cyp2b1 and Cyp4a1 when compared to the controls. Regarding the phase II enzyme gene transcripts, Ugt1a6, Ugt2b1 and Ephx1 were up-regulated when compared to the controls.

In the pituitary gland, Tshb gene transcripts were slightly up-regulated (not statistically significant) when compared to the controls.

Also at 1500 ppm PROD activity was increased by 1.3-fold when compared to the controls and phase I enzyme Cyp2b1 and Cyp4a1 gene transcripts were up-regulated. Regarding the phase II enzyme gene transcripts, Ugt1a6 and Ephx1 were up-regulated when compared to the controls. Ugt2b1 gene transcripts were very slightly up-regulated ((not statistically significant) when compared to the controls. In the pituitary gland, Tshb gene transcripts were very slightly up-regulated (not statistically significant) when compared to the controls.

This study was not performed according to any OECD guideline on male rats. Only brain and liver were weighed, not the thyroid. Liver enzyme induction can perhaps explain some of the thyroid effects but it can not exclude the presence of other MoA for thyroid effects. Hence, the question of MoA is not considered to be explained thoroughly.

Desmedipham treatment up-regulates Ugt2b1 transcript from the UDP glucuronosyltransferase gene family in rats.

Description of key information

Conclusion:
The evaluation of the results of this study indicates that the thyroid effects can be explained as a consequence of liver enzyme induction because some evidence of CAR activation was observed qualitatively through increased PROD activity and induction of Phase II enzymes (Ugt1a6, Ugt2b1 transcripts; T4-GT increased) associated with an increase of TSH (Tshb transcript increased). Although the magnitude of changes of key parameters investigated in this study was not very high such MOA for inducing thyroid effects in rodents is not considered relevant for humans.

Test method/  species	Result	Assessment	Reference
- Mechanistic	Not specified.	Supporting study	Blanck (2014)